Surface display of antigens on gram-negative outer membrane vesicles

ABSTRACT

The present invention relates to vaccine compositions based on Gram-negative outer membrane vesicles displaying antigens of pathogens expressed as part of a fusion protein comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria, and use of such compositions in vaccination. The invention further relates to the fusion lipoproteins comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria and antigens of pathogens fused thereto, DNA constructs and bacterial host cells for expressing these fusion lipoproteins and to methods for producing outer membrane vesicles displaying the fusion lipoproteins.

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particularthe fields of vaccinology, medical microbiology, bacteriology andimmunology. More specifically, the invention relates to vaccinecomposition based on Gram-negative outer membrane vesicles displayingantigens of pathogens, preferably Borrelia antigens, and use of thesecompositions in vaccination.

BACKGROUND OF THE INVENTION

Outer Membrane Vesicles (OMVs) are spherical buddings of the outermembrane (OM) that are spontaneously produced by Gram-negative bacteria.They are composed of OM proteins, LPS, phospholipids, and entrappedperiplasmic components. Because of their excellent immunostimulatoryproperties (1-3) and ease of production, OMVs are receiving more andmore attention as vaccine candidates. Immunization studies in mice havedemonstrated that OMVs can protect against challenges with variouspathogenic bacteria (4-12). For Neisseria meningitidis, OMV vaccineshave been extensively investigated in clinical trials, and two OMV-basedvaccines against Neisseria (MenBvac and MeNZB) are already available forhuman use (13, 14).

Because of their intrinsic adjuvant properties, the use of OMVs as adelivery vehicle for heterologous antigens has gained considerableinterest (15). Several studies have demonstrated that the expression ofheterologous antigens in the periplasm or OM of Gram-negative bacteria,by fusion of the heterologous protein to signal peptides or carrierproteins of the host, can lead to their inclusion in OMVs (1-3, 16-19).Importantly, such recombinant OMVs can induce an immune response to theheterologous antigen in immunized mice (2, 3, 12, 17, 18), and evenprotect them against an otherwise lethal challenge with the pathogenfrom which the antigen originates (3, 17).

To what extent the specific location of a heterologous antigen withinthe OMV (periplasm/inside of OM/outside of OM) affects the immuneresponse remains an open question. Theoretically, the outer surface ofthe OMV appears to be the best option, as this provides the bestaccessibility for the binding of B-cell receptors (17). There is indeedaccumulating evidence that surface exposed antigens evoke superiorimmune responses (20-24), which makes the precise targeting ofheterologous antigens to the OMV surface of special interest.

Various expression systems that specifically target the expression ofheterologous proteins to the outer surface of bacterial cells have beendeveloped (see (25-29) for reviews). However, many of these systems canonly display small parts of proteins and suffer from low expressionlevels (30). The two most versatile approaches fuse (parts of)heterologous proteins to Ice Nucleation Protein (25, 31) orautotransporters (21, 32-35) to reach the cell surface. Recently, bothsystems have also been used to decorate the surface of OMVs withmultiple enzymes/antigens (21, 31).

Lipoproteins are membrane-bound proteins that are emerging as keytargets for protective immunity, because of their excellentimmunostimulatory properties and role as virulence factors. For example,OspA (Borrelia burgdorferi) and fHbp (N. meningitidis) have both beenextensively studied as vaccine components against Lyme disease (36-39)and meningitis (40, 41), respectively. Surface expression ofheterologous lipoproteins in OMVs has however not been explored so far.

Lipoproteins carry a lipid-modification on their N-terminal cysteine,facilitating the anchoring of hydrophilic proteins in hydrophobicmembranes. This highly conserved protein lipidation motif is recognizedby the mammalian innate immune system through the Toll like receptorTLR2, providing lipoproteins with superior immunostimulatory properties(44, 45). In Gram-negative bacteria, most lipoproteins are found on theperiplasmic side of the inner or outer membrane. They are transferredfrom the inner membrane to the outer membrane by the Lol (localizationof lipoproteins) machinery (46). Lipoproteins that are located on theextracellular side of the outer membrane are less common, and systems orsignals guiding transfer over the outer membrane have not yet beenelucidated.

In Borrelia, lipoproteins seem to be transferred to the outside of theouter membrane by default, so that the surface of this spirochete isunusually rich in lipoproteins (47). One example of a Borrelialipoprotein with a surface localization is OspA, for which detailedknowledge regarding its immunogenicity and structure is availablebecause OspA has been extensively investigated as a vaccine componentagainst Lyme disease.

Lyme disease is the most common vector-borne disease in Europe and theUnited States. Lyme disease is a multisystemic inflammatory disorderthat is caused by infection with spirochetes of the B. burgdorferi sensulato complex as a result of a bite by infected ticks. If an infection isnot treated with antibiotics, it can eventually develop into a chronicdisease with severe pathology. The only vaccine shortly available forhuman use (Lymerix) was based on recombinant lipidated OspA. Due to poorsales resulting from claims about auto-immune side-effects, this vaccinewas voluntarily withdrawn from the market in 2002, only three yearsafter its introduction (49). However, the side-effect claims were laterfound to be unsubstantiated (50) and recent Lyme vaccine developmentsstill target OspA, with the much-disputed epitope removed (38, 39).

There is however, still a need in the art for improved vaccinecompositions based on Gram-negative outer membrane vesicles displayingantigens of pathogens at their surface, such as Borrelia antigens, anduse of these compositions in vaccination e.g. against Lyme disease.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a fusion lipoproteincomprising an N-terminal and a C-terminal fusion partner, wherein: a)the N-terminal fusion partner comprises in N- to C-terminal order: i) alipidated N-terminal cysteine; ii) a tether of a surface exposedlipoprotein of a Gram-negative bacterium, wherein preferably the tetheris located adjacent to the lipidated N-terminal cysteine; and,preferably, iii) a stretch of at least 5, 10, or 17 contiguous aminoacids that are located C-terminally of a tether in the amino acidssequence of a surface exposed lipoprotein of a Gram-negative bacterium;and wherein the N-terminal fusion partner causes expression of thefusion lipoprotein on the extracellular outermembrane surface of aGram-negative bacterium upon expression therein; and, b) the C-terminalfusion partner comprises at least one epitope of an antigen associatedwith an infectious disease and/or a tumour, and wherein, preferably, theamino acid sequence of the fusion lipoprotein does not occur in nature.Preferably, in the fusion lipoprotein, the N-terminal fusion partnercomprises an N-terminal fragment from a surface exposed lipoprotein of aGram-negative bacterium and wherein the fragment causes surfaceexpression of the fusion lipoprotein when expressed in the Gram-negativebacterium. Preferably, the N-terminal fragment is from a surface exposedlipoprotein of a Gram-negative bacterium of the genus Neisseria,preferably a Neisseria meningitidis, Neisseria gonorrhoeae or N.lactamica, and more preferably the surface exposed lipoprotein isselected from the group consisting of fHbp, LpbB, TbpB, HpuA, NHBA andAg473.

In a preferred fusion lipoprotein according to the invention, theN-terminal fusion partner at least comprises: a) an amino acid sequencethat has at least 60% sequence identity to the amino acid sequence inpositions 20-38 of SEQ ID NO: 1, preferably an amino acid sequence thathas at least 60% sequence identity to the amino acid sequence inpositions 20-50 of SEQ ID NO: 1; b) an amino acid sequence that has atleast 60% sequence identity to the amino acid sequence in positions21-61 or positions 21-63 of SEQ ID NO: 2, preferably an amino acidsequence that has at least 60% sequence identity to the amino acidsequence in positions 21-73 or positions 21-75 of SEQ ID NO: 2; or, c)an amino acid sequence that has at least 60% sequence identity to theamino acid sequence in positions 23-51 of SEQ ID NO: 3, preferably anamino acid sequence that has at least 60% sequence identity to the aminoacid sequence in positions 23-63 of SEQ ID NO: 3.

Preferably, in a fusion lipoprotein according to the invention, theC-terminal fusion partner lacks amino acid sequences from a surfaceexposed lipoprotein from which the sequences of the N-terminal fusionpartner are derived.

The C-terminal fusion partner in a fusion lipoprotein of the invention,preferably, comprises or consists of surface exposed epitopes from aproteinaceous antigen of an infectious agent or tumour. More preferably,the C-terminal fusion partner comprises or consists of a surface exposeddomain of a surface exposed bacterial protein or lipoprotein. Thesurface exposed bacterial protein or lipoprotein preferably is aBorrelia surface lipoprotein, preferably selected from the groupconsisting of OspA, OspB, OspC, OspF, VlsE, BbCRASP1, Vsp1, P35 (BBK32),P37 (BBK50), P39, P66, DpbA and BB017. More preferably, the Borreliasurface lipoprotein comprises or consists of amino acids 29-273 of SEQID NO: 4 or amino acids 29-273 of SEQ ID NO: 58 or amino acids 136-210of SEQ ID NO: 59.

In a second aspect, the invention pertains to an OMV comprising a fusionlipoprotein of the invention, wherein the OMV preferably is not adetergent-extracted OMV. Suitable OMV that are not detergent-extractedare supernatant OMV or native OMV, wherein preferably the OMV is anative OMV.

An OMV comprising a fusion lipoprotein of the invention, preferably isobtained/obtainable from a Gram-negative bacterium that has one or moregenetic modifications selected from the group consisting of: a) agenetic modification causing the bacterium to produce an LPS withreduced toxicity, wherein preferably the genetic modification reduces oreliminates expression of at least one of a lpxL1, lpxL2 and lpxK gene ora homologue thereof and/or increases the expression of at least one of alpxE, lpxF and pagL genes; b) genetic modification that increasesvesicle formation, wherein preferably, the genetic modification reducesor eliminates expression of an ompA gene or homologue thereof, morepreferably a rmpM gene or homologue thereof; and, c) geneticmodification that prevent proteolytic release of cell surface-exposedlipoprotein, wherein preferably, the genetic modification reduces oreliminates expression of a nalP gene or homologue thereof. TheGram-negative bacterium where the OMV of the invention are producedpreferably belongs to a genus selected from the group consisting ofNeisseria, Bordetella, Escherichia and Salmonella, more preferably thebacterium belongs to a species selected from the group consisting ofNeisseria meningitidis, Bordetella pertussis, Escherichia coli andSalmonella enterica.

In a third aspect, the invention relates to a pharmaceutical compositioncomprising an OMV of the invention and a pharmaceutically acceptedexcipient.

In a fourth aspect, the invention relates to an OMV according to of theinvention, or a pharmaceutical composition comprising the OMV, for useas a medicament.

In a fifth aspect, the invention relates to an OMV according to of theinvention, or a pharmaceutical composition comprising the OMV, for theprevention or treatment of an infectious disease or tumour associatedwith the antigen, wherein preferably the infectious disease is aBorrelia infection, more preferably a Borrelia burgdorferi infection.

In a sixth aspect, the invention relates to a nucleic acid moleculeencoding a pre-profusion lipoprotein, wherein upon expression in aGram-negative bacterium the pre-profusion lipoprotein matures into thefusion lipoprotein of the invention, and wherein preferably the nucleicacid molecule is an expression construct for expression of thepre-profusion lipoprotein in a Gram-negative bacterium.

In a seventh aspect, the invention relates to a Gram-negative bacterialhost cell comprising a nucleic acid molecule or an expression constructcomprising a nucleic acid sequence encoding the pre-profusionlipoprotein, wherein preferably the Gram-negative bacterium belongs to agenus selected from the group consisting of Neisseria, Bordetella,Escherichia and Salmonella, more preferably the bacterium belongs to aspecies selected from the group consisting of Neisseria meningitidis,Bordetella pertussis, Escherichia coli and Salmonella enterica.

In an eighth aspect, the invention relates to a method for producing anOMV comprising a fusion lipoprotein of the invention, wherein the methodcomprises the steps of: i) cultivating Gram-negative bacterial host cellcomprising a nucleic acid molecule or an expression construct comprisinga nucleic acid sequence encoding the pre-profusion lipoprotein; ii)optionally extracting the OMV; and, iii) recovering the OMV, wherein therecovery at least comprises removal of the bacteria from the OMV, andwherein preferably, the method is detergent-free.

DESCRIPTION OF THE INVENTION

Definitions

The terms “homology”, “sequence identity” and the like are usedinterchangeably herein. Sequence identity is herein defined as arelationship between two or more amino acid (polypeptide or protein)sequences or two or more nucleic acid (polynucleotide) sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between amino acid or nucleic acidsequences, as the case may be, as determined by the match betweenstrings of such sequences. “Similarity” between two amino acid sequencesis determined by comparing the amino acid sequence and its conservedamino acid substitutes of one polypeptide to the sequence of a secondpolypeptide. “Identity” and “similarity” can be readily calculated byknown methods.

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms, depending on the length of the twosequences. Sequences of similar lengths are preferably aligned using aglobal alignment algorithms (e.g. Needleman Wunsch) which aligns thesequences optimally over the entire length, while sequences ofsubstantially different lengths are preferably aligned using a localalignment algorithm (e.g. Smith Waterman). Sequences may then bereferred to as “substantially identical” or “essentially similar” whenthey (when optimally aligned by for example the programs GAP or BESTFITusing default parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength (full length), maximizing the number of matches and minimizingthe number of gaps. A global alignment is suitably used to determinesequence identity when the two sequences have similar lengths.Generally, the GAP default parameters are used, with a gap creationpenalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3(nucleotides)/2 (proteins). For nucleotides the default scoring matrixused is nwsgapdna and for proteins the default scoring matrix isBlosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequencealignments and scores for percentage sequence identity may be determinedusing computer programs, such as the GCG Wisconsin Package, Version10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego,Calif. 92121-3752 USA, or using open source software, such as theprogram “needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in EmbossWIN version2.10.0, using the same parameters as for GAP above, or using the defaultsettings (both for ‘needle’ and for ‘water’ and both for protein and forDNA alignments, the default Gap opening penalty is 10.0 and the defaultgap extension penalty is 0.5; default scoring matrices are Blossum62 forproteins and DNAFull for DNA). When sequences have a substantiallydifferent overall lengths, local alignments, such as those using theSmith Waterman algorithm, are preferred.

Alternatively percentage similarity or identity may be determined bysearching against public databases, using algorithms such as FASTA,BLAST, etc. Thus, the nucleic acid and protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify other family membersor related sequences. Such searches can be performed using the BLASTnand BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to oxidoreductase nucleic acid molecules of the invention.BLAST protein searches can be performed with the BLASTx program,score=50, wordlength=3 to obtain amino acid sequences homologous toprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., BLASTx and BLASTn) can be used. See thehomepage of the National Center for Biotechnology Information athttp://www.ncbi.nlm.nih.gov/.

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagines and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

As used herein, the term “selectively hybridizing”, “hybridizesselectively” and similar terms are intended to describe conditions forhybridization and washing under which nucleotide sequences at least 66%,at least 70%, at least 75%, at least 80%, more preferably at least 85%,even more preferably at least 90%, preferably at least 95%, morepreferably at least 98% or more preferably at least 99% homologous toeach other typically remain hybridized to each other. That is to say,such hybridizing sequences may share at least 45%, at least 50%, atleast 55%, at least 60%, at least 65, at least 70%, at least 75%, atleast 80%, more preferably at least 85%, even more preferably at least90%, more preferably at least 95%, more preferably at least 98% or morepreferably at least 99% sequence identity.

A preferred, non-limiting example of such hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 1×SSC, 0.1% SDS at about 50° C.,preferably at about 55° C., preferably at about 60° C. and even morepreferably at about 65° C.

Highly stringent conditions include, for example, hybridization at about68° C. in 5× SSC/5× Denhardt's solution/1.0% SDS and washing in0.2×SSC/0.1% SDS at room temperature. Alternatively, washing may beperformed at 42° C.

The skilled artisan will know which conditions to apply for stringentand highly stringent hybridization conditions. Additional guidanceregarding such conditions is readily available in the art, for example,in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.; and Ausubel et al. (eds.), Sambrook andRussell (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork 1995, Current Protocols in Molecular Biology, (John Wiley & Sons,N.Y.).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of mRNAs), or to a complementarystretch of T (or U) resides, would not be included in a polynucleotideof the invention used to specifically hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

A “nucleic acid construct” or “nucleic acid vector” is herein understoodto mean a man-made nucleic acid molecule resulting from the use ofrecombinant DNA technology.

The term “nucleic acid construct” therefore does not include naturallyoccurring nucleic acid molecules although a nucleic acid construct maycomprise (parts of) naturally occurring nucleic acid molecules. Theterms “expression vector” or “expression construct” refer to nucleotidesequences that are capable of effecting expression of a gene in hostcells or host organisms compatible with such sequences. These expressionvectors typically include at least suitable transcription regulatorysequences and optionally, 3′ transcription termination signals.Additional factors necessary or helpful in effecting expression may alsobe present, such as expression enhancer elements. The expression vectorwill be introduced into a suitable host cell and be able to effectexpression of the coding sequence in an in vitro cell culture of thehost cell. The expression vector will be suitable for replication in thehost cell or organism of the invention.

As used herein, the term “promoter” or “transcription regulatorysequence” refers to a nucleic acid fragment that functions to controlthe transcription of one or more coding sequences, and is locatedupstream with respect to the direction of transcription of thetranscription initiation site of the coding sequence, and isstructurally identified by the presence of a binding site forDNA-dependent RNA polymerase, transcription initiation sites and anyother DNA sequences, including, but not limited to transcription factorbinding sites, repressor and activator protein binding sites, and anyother sequences of nucleotides known to one of skill in the art to actdirectly or indirectly to regulate the amount of transcription from thepromoter. A “constitutive” promoter is a promoter that is active in mosttissues under most physiological and developmental conditions. An“inducible” promoter is a promoter that is physiologically ordevelopmentally regulated, e.g. by the application of a chemicalinducer.

The term “selectable marker” is a term familiar to one of ordinary skillin the art and is used herein to describe any genetic entity which, whenexpressed, can be used to select for a cell or cells containing theselectable marker. The term “reporter” may be used interchangeably withmarker, although it is mainly used to refer to visible markers, such asgreen fluorescent protein (GFP). Selectable markers may be dominant orrecessive or bidirectional.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a transcription regulatorysequence is operably linked to a coding sequence if it affects thetranscription of the coding sequence. Operably linked means that the DNAsequences being linked are typically contiguous and, where necessary tojoin two protein encoding regions, contiguous and in reading frame.

The term “peptide” as used herein is defined as a chain of amino acidresidues, usually having a defined sequence. As used herein the termpeptide is interchangeable with the terms “polypeptide” and “protein”.In the context of the present invention, the term “peptide” is definedas being any peptide or protein comprising at least two amino acidslinked by a modified or unmodified peptide bond. The term “peptide”refers to short-chain molecules such as oligopeptides or oligomers or tolong-chain molecules such as proteins. A protein/peptide can be linear,branched or cyclic. The peptide can include D amino acids, L aminoacids, or a combination thereof. A peptide according to the presentinvention can comprise modified amino acids. Thus, the peptide of thepresent invention can also be modified by natural processes such aspost-transcriptional modifications or by a chemical process. Someexamples of these modifications are: acetylation, acylation,ADP-ribosylation, amidation, covalent bonding with flavine, covalentbonding with a heme, covalent bonding with a nucleotide or a nucleotidederivative, covalent bonding to a modified or unmodified carbohydratemoiety, bonding with a lipid or a lipid derivative, covalent bondingwith a phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, cysteine molecule formation, pyroglutamateformation, formylation, gamma-carboxylation, hydroxylation, iodination,methylation, oxidation, phosphorylation, racemization, hydroxylation,etc. Thus, any modification of the peptide which does not have theeffect of eliminating the immunogenicity of the peptide, is coveredwithin the scope of the present invention.

The term “gene” means a DNA fragment comprising a region (transcribedregion), which is transcribed into an RNA molecule (e.g. an mRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene will usually comprise several operably linked fragments, such asa promoter, a 5′ leader sequence, a coding region and a 3′-nontranslatedsequence (3′-end) comprising a polyadenylation site. “Expression of agene” refers to the process wherein a DNA region which is operablylinked to appropriate regulatory regions, particularly a promoter, istranscribed into an RNA, which is biologically active, i.e. which iscapable of being translated into a biologically active protein orpeptide. The term “homologous” when used to indicate the relationbetween a given (recombinant) nucleic acid or polypeptide molecule and agiven host organism or host cell, is understood to mean that in naturethe nucleic acid or polypeptide molecule is produced by a host cell ororganisms of the same species, preferably of the same variety or strain.If homologous to a host cell, a nucleic acid sequence encoding apolypeptide will typically (but not necessarily) be operably linked toanother (heterologous) promoter sequence and, if applicable, another(heterologous) secretory signal sequence and/or terminator sequence thanin its natural environment. It is understood that the regulatorysequences, signal sequences, terminator sequences, etc. may also behomologous to the host cell. In this context, the use of only“homologous” sequence elements allows the construction of “self-cloned”genetically modified organisms (GMO's) (self-cloning is defined hereinas in European Directive 98/81/EC Annex II). When used to indicate therelatedness of two nucleic acid sequences the term “homologous” meansthat one single-stranded nucleic acid sequence may hybridize to acomplementary single-stranded nucleic acid sequence. The degree ofhybridization may depend on a number of factors including the amount ofidentity between the sequences and the hybridization conditions such astemperature and salt concentration as discussed later.

The terms “heterologous” and “exogenous” when used with respect to anucleic acid (DNA or RNA) or protein refers to a nucleic acid or proteinthat does not occur naturally as part of the organism, cell, genome orDNA or RNA sequence in which it is present, or that is found in a cellor location or locations in the genome or DNA or RNA sequence thatdiffer from that in which it is found in nature. Heterologous andexogenous nucleic acids or proteins are not endogenous to the cell intowhich it is introduced, but have been obtained from another cell orsynthetically or recombinantly produced. Generally, though notnecessarily, such nucleic acids encode proteins, i.e. exogenousproteins, that are not normally produced by the cell in which the DNA istranscribed or expressed. Similarly exogenous RNA encodes for proteinsnot normally expressed in the cell in which the exogenous RNA ispresent. Heterologous/exogenous nucleic acids and proteins may also bereferred to as foreign nucleic acids or proteins. Any nucleic acid orprotein that one of skill in the art would recognize as foreign to thecell in which it is expressed is herein encompassed by the termheterologous or exogenous nucleic acid or protein. The termsheterologous and exogenous also apply to non-natural combinations ofnucleic acid or amino acid sequences, i.e. combinations where at leasttwo of the combined sequences are foreign with respect to each other.

The term “immune response” as used herein refers to the production ofantibodies and/or cells (such as T lymphocytes) that are directedagainst, and/or assist in the decomposition and/or inhibition of, aparticular antigenic entity, carrying and/or expressing or presentingantigens and/or antigenic epitopes at its surface. The phrases “aneffective immunoprotective response”, “immunoprotection”, and liketerms, for purposes of the present invention, mean an immune responsethat is directed against one or more antigenic epitopes of a pathogen, apathogen-infected cell or a cancer cell so as to protect againstinfection by the pathogen or against cancer in a vaccinated subject. Forpurposes of the present invention, protection against infection by apathogen or protection against cancer includes not only the absoluteprevention of infection or cancer, but also any detectable reduction inthe degree or rate of infection by a pathogen or of the cancer, or anydetectable reduction in the severity of the disease or any symptom orcondition resulting from infection by the pathogen or cancer in thevaccinated subject, for example as compared to an unvaccinated infectedsubject. An effective immunoprotective response in the case of canceralso includes clearing up the cancer cells, thereby reducing the size ofcancer or even abolishing the cancer. Vaccination in order to achievethis is also called therapeutic vaccination. Alternatively, an effectiveimmunoprotective response can be induced in subjects that have notpreviously been infected with the pathogen and/or are not infected withthe pathogen or do not yet suffer from cancer at the time ofvaccination, such vaccination can be referred to as prophylacticvaccination.

According to the present invention, the general use herein of the term“antigen” refers to any molecule that binds specifically to an antibody.The term also refers to any molecule or molecular fragment that can bebound by an MHC molecule and presented to a T-cell receptor. Antigenscan be e.g. proteinaceous molecules, i.e. polyaminoacid sequences,optionally comprising non-protein groups such as carbohydrate moietiesand/or lipid moieties or antigens can be e.g. molecules that are notproteinaceous such as carbohydrates. An antigen can be e.g. any portionof a protein (peptide, partial protein, full-length protein), whereinthe protein is naturally occurring or synthetically derived, a cellularcomposition (whole cell, cell lysate or disrupted cells), an organism(whole organism, lysate or disrupted cells) or a carbohydrate or othermolecule, or a portion thereof, that is able to elicit anantigen-specific immune response (humoral and/or cellular immuneresponse) in a particular subject, which immune response preferably ismeasurable via an assay or method.

The term “antigen” is herein understood as a structural substance whichserves as a target for the receptors of an adaptive immune response. Anantigen thus serves as target for a TCR (T-cell receptor) or a BCR(B-cell receptor) or the secreted form of a BCR, i.e. an antibody. Theantigen can thus be a protein, peptide, carbohydrate or other haptenthat is usually part of a larger structure, such as e.g. a cell or avirion. The antigen may originate from within the body (“self”) or fromthe external environment (“non-self”). The immune system is usuallynon-reactive against “self” antigens under normal conditions due tonegative selection of T cells in the thymus and is supposed to identifyand attack only “non-self” invaders from the outside world ormodified/harmful substances present in the body under e.g. diseaseconditions. Antigens structures that are the target of a cellular immuneresponse are presented by antigen presenting cells (APC) in the form ofprocessed antigenic peptides to the T cells of the adaptive immunesystem via a histocompatibility molecule. Depending on the antigenpresented and the type of the histocompatibility molecule, several typesof T cells can become activated. For T-Cell Receptor (TCR) recognition,the antigen is processed into small peptide fragments inside the celland presented to a T-cell receptor by major histocompatibility complex(MHC). The term “immunogen” is used herein to describe an entity thatcomprises or encodes at least one epitope of an antigen such that whenadministered to a subject, preferably together with an appropriateadjuvant, elicits a specific humoral and/or cellular immune response inthe subject against the epitope and antigen comprising the epitope. Animmunogen can be identical to the antigen or at least comprises a partof the antigen, e.g. a part comprising an epitope of the antigen.Therefore, to vaccinate a subject against a particular antigen means, inone embodiment, that an immune response is elicited against the antigenor immunogenic portion thereof, as a result of administration of animmunogen comprising at least one epitope of the antigen. Vaccinationpreferably results in a protective or therapeutic effect, whereinsubsequent exposure to the antigen (or a source of the antigen) elicitsan immune response against the antigen (or source) that reduces orprevents a disease or condition in the subject. The concept ofvaccination is well-known in the art. The immune response that iselicited by administration of a prophylactic or therapeutic compositionof the present invention can be any detectable change in any facet ofthe immune status (e.g., cellular response, humoral response, cytokineproduction), as compared to in the absence of the administration of thevaccine.

An “epitope” is defined herein as a single immunogenic site within agiven antigen that is sufficient to elicit an immune response in asubject. Those of skill in the art will recognize that T cell epitopesare different in size and composition from B cell epitopes, and that Tcell epitopes presented through the Class I MHC pathway differ fromepitopes presented through the Class II MHC pathway. Epitopes can belinear sequences or conformational epitopes (conserved binding regions)depending on the type of immune response. An antigen can be as small asa single epitope, or larger, and can include multiple epitopes. As such,the size of an antigen can be as small as about 5-12 amino acids (e.g.,a peptide) and as large as: a full length protein, including multimericproteins, protein complexes, virions, particles, whole cells, wholemicroorganisms, or portions thereof (e.g., lysates of whole cells orextracts of microorganisms).

OMV (also referred to as “blebs”) are bi-layered membrane structures,usually spherical, with a diameter in the range of 20-250 nm (sometimes10-500 nm), that are pinched off from the outer membrane ofGram-negative bacteria. The OMV membrane contains phospholipids (PL) onthe inside and lipopolysaccharides (LPS) and PL on the outside, mixedwith membrane proteins in various positions, largely reflecting thestructure of the bacterial outer membrane from which they pinched off.The lumen of the OMV may contain various compounds from the periplasm orcytoplasm, such as proteins, RNA/DNA, and peptidoglycan (PG), however,unlike bacterial cells, OMV lack the ability to self-replicate. In thecontext of the present invention three type of OMV can be distinguisheddepending on the method of their production. sOMV are spontaneous ornatural OMV, that are purified and concentrated from culturesupernatant, by separating intact cells from the already formed OMVs.Detergent OMV, dOMV, are extracted from cells with detergent, such asdeoxycho late, which also reduces the content of reactogenic LPS and oflipoproteins. After detergent extraction dOMV are separated from cellsand cellular debris and further purified and concentrated. Finally, theterm native nOMV is used herein for OMV that are generated fromconcentrated dead cells with non-detergent cell disruption techniques,or that are extracted from cells with other (non-disruptive)detergent-free methods, to be able to clearly distinguish them from thewild-type spontaneous OMVs and from the detergent-extracted dOMV.

Any reference to nucleotide or amino acid sequences accessible in publicsequence databases herein refers to the version of the sequence entry asavailable on the filing date of this document.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Gram-negative OMVs comprising surfaceexposed fusion lipoprotein comprising epitopes of antigens forvaccination purposes. Surface lipoproteins are normally removed from theOMV during conventional detergent-based removal of LPS. However, recentbiotechnological developments have led to detergent-free OMV extractionprocesses, e.g. from in Neisseria, that would potentially allow forsurface exposed lipoproteins to remain attached to the OMV (42, 43). Thepresent inventors have investigated the possibility for surfaceexpression of antigenic lipoproteins in these so-called native OMVs(nOMVs) including heterologous lipoproteins. Specifically the inventorshave tested the heterologous expression of the Borrelia OspA lipoproteinin Neisseria nOMVs. Because of its surface localization in Borrelia andthe detailed knowledge regarding its immunogenicity and structure, OspAwould be a suitable lipoprotein to test.

Even though the inventors were able to express OspA in N. meningitidiscells and nOMVs, they were unable to detect it on the meningococcal cellsurface. This indicates mislocalization to the periplasm or theperiplasmic side of the OM. Such host-switch induced mislocalization oflipoproteins is not uncommon and probably results from adherence to thesurrogate host's sorting rules (51).

Surprisingly we were able to redirect OspA to the cell surface ofNeisseria, by fusing its globular domain to different parts of fHbp, awell-studied meningococcal surface lipoprotein (41, 48). We show thatfusion to specific N-terminal parts of fHbp allows surface expression ofthe fHbp-OspA fusion constructs. Moreover, we demonstrate that NeisserianOMV expressing these surface-exposed fHbp-OspA hybrids elicit strongantibody responses in immunized mice.

Secondly, we were also able to redirect OspA to the cell surface ofNeisseria, by fusing its globular domain to different parts oftransferrin binding protein B (TbpB), which has been well characterizedat the structural level and which is a co-receptor involved in ironpiracy (63).

Thirdly, we were able to redirect other proteins, such as OspC and RmpM,to the cell surface of Neisseria, including non-borrelial andnon-liporoteins (such as RmpM). In a first aspect the invention pertainsto a fusion lipoprotein. The fusion lipoprotein preferably at leastcomprises an N-terminal and a C-terminal fusion partner.

The N-terminal fusion partner in the fusion lipoprotein is intended toeffect expression of the fusion protein on the extracellular surface ofthe outermembrane of the Gram-negative bacterium wherein the fusionprotein is expressed as well as anchoring into that membrane through itscovalently attached lipid. To this end, the N-terminal fusion partner inthe fusion lipoprotein preferably at least comprises, preferably in N-to C-terminal order: i) a lipidated N-terminal cysteine; ii) a tether ofa surface exposed lipoprotein of a Gram-negative bacterium, whereinpreferably the tether is located (immediately) adjacent to the lipidatedN-terminal cysteine; and, preferably, iii) at least 1 or a stretch of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 25, 28 or 30 contiguous amino acids that are located immediatelyC-terminal of a tether in the amino acids sequence of a surface exposedlipoprotein of a Gram-negative bacterium.

The N-terminal fusion partner preferably causes surface expression ofthe fusion lipoprotein on the extracellular outermembrane surface whenexpressed in a Gram-negative bacterium. The ability of the N-terminalfusion partner to cause such surface expression of the fusionlipoprotein can be assayed by expression of the fusion lipoprotein in aGram-negative bacterium and detection of the fusion lipoprotein on theoutside of the Gram-negative bacterium, e.g. using an antibody againstthe fusion lipoprotein, preferably an antibody against the C-terminalfusion partner, whereby the bound antibody preferably is detected byimmunofluorescence, e.g. as described in Examples 1.5 and 2.3 herein. Ina preferred assay for determining the ability of the N-terminal fusionpartner to cause surface expression, the N-terminal fusion partner isfused to a C-terminal fusion partner that is known to be capable ofsurface expression as (part of) a lipoprotein on the extracellularoutermembrane surface of a Gram-negative bacterium.

A suitable C-terminal fusion partner for this purpose is e.g. theglobular domain of a Borrelia OspA lipoprotein, preferably the globulardomain of a Borrelia burgdorferi OspA lipoprotein, whereby preferablythe globular domain consists of the amino acid sequence of positions29-273 of an OspA lipoprotein, e.g. the amino acid sequence of positions29-273 of SEQ ID NO: 4, as used in the Examples herein.

An alternative suitable C-terminal fusion partner for testing theability of the N-terminal fusion partner to cause surface expression,comprises or consists e.g. of the globular domain of a Borrelia afzeliiOspA lipoprotein, whereby preferably the globular domain consists ofe.g. the amino acid sequence of positions 29-273 of SEQ ID NO: 58, asused in the Examples herein.

Another alternative suitable C-terminal fusion partner for testing theability of the N-terminal fusion partner to cause surface expression,comprises or consists e.g. of (a fragment of) the globular domain of aBorrelia OspC lipoprotein, preferably a B. burgdorferi OspC lipoprotein,whereby preferably the fragment consists of the amino acid sequence ofpositions 136-210 of an OspC lipoprotein, e.g. the amino acid sequenceof positions 136-210 of SEQ ID NO: 59, as used in the Examples herein.

Alternatively, the C-terminal fusion partner for testing the ability ofthe N-terminal fusion partner to cause surface expression, preferablycomprises or consists of a domain of a periplasmic bacterial protein.Preferably the domain of a periplasmic bacterial protein is from abacterium selected from a genus consisting of Bordetella, Borrelia,Coxiella, Neisseria and any of the other pathogenic bacterial generamentioned above. The domain of the periplasmic bacterial proteinpreferably associates with peptidoglycan and/or preferably is theC-terminal domain of the protein. More preferably, the domain of theperiplasmic Neisseria protein is derived from RmpM, more preferably theNeisseria periplasmic protein comprises or consists of amino acids90-242 of SEQ ID NO: 7, as used in the Examples herein.

In a preferred assay for the determining the ability of the N-terminalfusion partner to cause surface expression, the fusion lipoprotein isexpressed in a Gram-negative bacterial host cell that is of the samespecies as the bacterium from which the majority of the sequences in theN-terminal fusion partner are obtained/obtainable, i.e. the bacteriumcontributing the highest number of individual amino acids to theN-terminal fusion partner. Thus, preferably the N-terminal fusionpartner at least causes expression on the extracellular outermembranesurface of a Gram-negative bacterium of a fusion lipoprotein consistingof the N-terminal fusion partner fused (at its C-terminus) to the aminoacid sequence of positions 29-273 of SEQ ID NO: 4 (or alternatively tothe amino acid sequence of positions 29-273 of SEQ ID NO: 58, or theamino acid sequence of positions 136-210 of SEQ ID NO: 59 or to theamino acid sequence of positions 90-242 of SEQ ID NO: 7), uponexpression in the Gram-negative bacterium, whereby the Gram-negativebacterium is of the same species as the bacterium from which themajority of the sequences in the N-terminal fusion partner areobtainable. Preferably, surface expression of this fusion lipoprotein isdetected by immunofluorescence microscopy with an anti-OspA (polyclonal)antibody, e.g. the anti-OspA (rabbit) antibody 200-401-C13S as availablefrom Rockland Immunochemicals Inc. (Limerick, Pa. 19468, USA;www.rockland-inc.com). Moreover, surface expression of the OspC fusionlipoprotein is preferably detected with an anti-OspC (polyclonal)antibody, e.g. the anti-OspC antibody 200-401-C11S as available fromRockland Immunochemicals Inc. The surface expression of the RmpM fusionlipoprotein is preferably detected with an anti-RmpM antibody MN2D6D asavailable from the National Institute for Public Health and theEnvironment, Bilthoven, the Netherlands.

The lipidated cysteine preferably is the most N-terminal amino acid inthe mature fusion lipoprotein of the invention. Bacterial lipoproteinsare initially translated as preprolipoproteins, which possess anN-terminal signal peptide of around 20 amino acids with typicalcharacteristic features of the signal peptides of secreted proteins(Inouye et al., 1977, PNAS USA 74:1004-1008). A conserved sequence atthe C-terminal region of the signal peptides, referred to as lipobox,[LVI] [ASTVI][GAS]C, is modified through the covalent attachment of adiacylglycerol moiety to the thiol group on the side chain of theindispensable cysteine residue (Babu et al., 2006, J. Bacteriol.188:2761-2773). This modification is catalyzed by the enzyme lipoproteindiacylglyceryl transferase, resulting in a prolipoprotein consisting ofa diacylglycerol moiety linked by a thioester bond to the protein. Theprolipoprotein is subsequently processed by the lipoprotein signalpeptidase, which cleaves off the signal peptide, leaving the lipidatedcysteine as the new N-terminal residue forming the mature lipoprotein.The mature lipoprotein can have an additional amide-linked fatty acidattached by a lipoprotein N-acyl transferase to the N-terminal cysteineresidue.

Downstream (in N- to C-terminal order) of the lipidated cysteine, theN-terminal fusion partner preferably comprises a tether of a surfaceexposed lipoprotein of a Gram-negative bacterium, whereby preferably thetether is located immediately adjacent to the N-terminal lipidatedcysteine, meaning that no additional amino acids are present between theN-terminal lipidated cysteine and the tether. Tethers of Gram-negativesurface lipoproteins are usually stretches of 5-50 amino acids with alow propensity of forming a secondary structure, such as an α-helix or aβ-strand or β-sheet, and which provide an unordered and flexiblelipopeptide tether to the remainder of the exposed structural protein.Without wishing to be bound by theory, the tether is further thought tobe important in determining the location of the lipoprotein, e.g.whether it is directed to the outer membrane by the lipoproteinlocalization machinery (Lol) or is retained at the inner membrane.Particularly the identity of the amino acid in position +2, i.e.immediately adjacent to the N-terminal lipidated cysteine has beenreported to be important for determining the location of thelipoprotein, even though this does not appear to be a universal rule andother amino acids more downstream in tether may also play a role inlocating the lipoprotein (Kovacs-Simon et al., 2011, Infect. Immun.79:548-561). Furthermore, as also shown by the present inventors, theability of a tether to effect surface expression of a lipoprotein can bespecies-specific. Preferably therefore, the tether in the fusion proteinis a tether from a surface expressed lipoprotein of a bacterial genus,more preferably of a bacterial species that is the same as the bacterialhost cell of the invention in which the fusion lipoprotein is expressed.The tether in the fusion lipoprotein is thus preferably homologous tothe host cell of the invention in which the fusion lipoprotein isexpressed.

Preferred tethers for expression of a fusion lipoprotein of theinvention in a Neisserial host cell are tethers from surface expressedNeisserial lipoproteins such as fHbp (factor H binding protein), LpbB(Lactoferrin binding protein), TbpB Transferrin binding protein), HpuA(hemoglobin-haptoglobin utilization protein), NHBA (Neisseria HeparinBinding Antigen, GNA2132) and Ag473 (Chu et al., 2012, PLoS One 7 (7):e40873; Genbank NP_274477.1) from a Neisseria such as e.g. N.meningitidis, N. gonorrhoeae and N. lactamica. A preferred tether forexpression of a fusion lipoprotein of the invention in a Neisserial hostcell is therefore a tether selected from the group consisting of a) anamino acid sequence that has at least 60, 69, 76, 84, 92 or 100%sequence identity to the amino acid sequence in positions 20-33 of SEQID NO: 1; b) an amino acid sequence that has at least 60, 68, 75, 81,87, 93 or 100% sequence identity to the amino acid sequence in positions21-56 or positions 21-58 of SEQ ID NO: 2 (e.g. derived from TbpB, strainMC58) or an amino acid sequence that has at least 60, 68, 75, 81, 87, 93or 100% sequence identity to the amino acid sequence in positions 21-56or positions 21-58 of SEQ ID NO: 60 (e.g. derived from TbpB, strainH44/76); and, c) an amino acid sequence that has at least 60, 66, 70,75, 79, 83, 87, 91, 95 or 100% sequence identity to the amino acidsequence in positions 23-46 of SEQ ID NO: 3, wherein preferably thetether has an amino acid sequence that naturally occurs in a surfaceexpressed Gram-negative lipoprotein. It is understood that theN-terminal lipidated cysteine is included in the definitions of theamino acid sequences of the tether in a), b) and c) above.

In a preferred embodiment, the N-terminal partner in a fusionlipoprotein of the invention comprises further sequences from a surfaceexposed lipoprotein of a Gram-negative bacterium. The inventors havefound that an N-terminal fusion partner comprising additional amino acidsequences of a surface exposed lipoprotein of a Gram-negative bacteriumcan significantly increase the level of surface expression of the fusionlipoprotein as e.g. exemplified by the fA1 fusion lipoprotein. TheN-terminal fusion partner therefore preferably comprises at least one ormore contiguous amino acids from an amino acids sequence of a surfaceexposed lipoprotein of a Gram-negative bacterium, wherein preferablythese one or more contiguous amino acids are present immediatelyC-terminal of a tether in the surface exposed lipoprotein from whichthey are derived. The length of this stretch of amino acids preferablyis as indicated above.

In a preferred embodiment the additional stretch of amino acids from asurface exposed lipoprotein comprise an amino acid sequence with apropensity to form a local element or segment of secondary structure, ora part thereof, such as an α-helix, a β-strand or a β-pleated sheet. Apreferred additional stretch of amino acids from a surface exposedlipoprotein that can be included for increasing the level of surfaceexpression of the fusion lipoprotein is a contiguous stretch of at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids takenfrom the amino acid sequence in positions 34-50 of a Neisserial fHbpprotein, more preferably of a N. meningitidis fHbp protein, mostpreferably contiguous stretch is taken from the amino acid sequence inpositions 34-50 of SEQ ID NO: 1. Preferably, amino acid in position 34is included in the contiguous stretch taken from the amino acid sequencein positions 34-50 of a Neisserial fHbp protein such that the stretch islocated immediately C-terminal of a tether in the amino acids sequenceof Neisserial fHbp protein.

In the N-terminal partner of a fusion lipoprotein of the invention thetether of a surface exposed lipoprotein of a Gram-negative bacterium andthe one or more contiguous amino acids from an amino acids sequence of asurface exposed lipoprotein of a Gram-negative bacterium, i.e. elementsii) and iii) above, can be obtained/obtainable from amino acid sequencesfrom two different surface exposed lipoproteins (that could even be fromtwo different Gram-negative bacteria), but preferably they areobtained/obtainable from an amino acid sequence of one and the samesurface exposed lipoprotein.

In one embodiment, in the fusion lipoprotein of the invention, theN-terminal fusion partner in the lipoprotein comprises an N-terminalfragment from a surface exposed lipoprotein of a Gram-negativebacterium, which N-terminal fragment at least includes the lipidatedcysteine. Preferably the N-terminal fusion partner in the lipoproteincomprises an N-terminal fragment from a mature surface exposedlipoprotein, wherein the mature lipoprotein is understood to have alipidated cysteine as N-terminus. Preferably, the N-terminal fusionpartner of the lipoprotein comprises at least 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 22, 25, 28, 30, 31, 32, 35, 38 or 40 contiguousamino acids from an N-terminal fragment of a mature surface exposedlipoprotein including and starting from the lipidated cysteine at theN-terminus of the fragment. Preferably, the N-terminal fragment causessurface expression of the fusion lipoprotein when expressed in theGram-negative bacterium. The ability of the N-terminal fragment toeffect surface expression of the fusion lipoprotein can be assayed asdescribed above.

In a preferred embodiment, the fusion lipoprotein of the invention is afusion lipoprotein that can be used for expression in a Neisserial hostcell. In the N-terminal partner of such a fusion lipoprotein,preferably, the tether of a surface exposed lipoprotein of aGram-negative bacterium and the one or more contiguous amino acids froman amino acids sequence of a surface exposed lipoprotein of aGram-negative bacterium, i.e. elements ii) and iii) above, and/or theN-terminal fragment as defined above, are obtained/obtainable from aminoacid sequences from a bacterium of the genus Neisseria, preferably aNeisseria meningitidis or Neisseria gonorrhoeae or N. lactamica. Morepreferably, the Neisserial surface exposed lipoprotein from which theamino acid sequences for the N-terminal fusion partner areobtained/obtainable is selected from the group consisting of fHbp, LpbB,TbpB, HpuA, NHBA and Ag473.

In another preferred embodiment, in a fusion lipoprotein of theinvention that can be used for expression in a Neisserial host cell, theN-terminal partner of the fusion lipoprotein comprises at least: a) anamino acid sequence that has at least 60% sequence identity to the aminoacid sequence in position 20 to one of positions 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 of SEQ ID NO: 1; b) anamino acid sequence that has at least 60% sequence identity to the aminoacid sequence in position 21 to one of positions 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 and 75 of SEQ ID NO:2; or, c) an amino acid sequence that has at least 60% sequence identityto the amino acid sequence in position 23 to one of positions 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 and 63 of SEQID NO: 3.

The C-terminal fusion partner in the fusion lipoprotein is preferablyintended to generate an immune response against at least one epitope ofan antigen associated with an infectious disease and/or a tumour, whichepitope is present in the C-terminal fusion partner. The C-terminalfusion partner in the fusion lipoprotein in principle can be any aminoacid sequence comprising the at least one epitope.

It is understood herein that a fusion lipoprotein of the inventionpreferably is a fusion protein wherein the N- and C-terminal fusionpartners are fused by normal protein synthesis in the Gram-negative hostcell wherein the fusion lipoprotein is expressed (see below) bytranslation of nucleic acid sequences coding for respectively the N- andC-terminal fusion partners, which coding sequences are operably linkedin frame by standard recombinant DNA techniques. Optionally, the N- andC-terminal fusion partners are fused through a linker amino acidsequence, for which the coding sequence is operably linked in frame withthe respective nucleic acid sequences coding for the N-and C-terminalfusion partners. The linker amino acid sequence preferably is amino acidsequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 amino acid. Apreferred linker comprises an amino acid sequence composed of the aminoacids glycine, proline, serine and alanine. More preferably, linkercomprises the amino acid sequence PGGSGA (SEQ ID NO: 5), or repeats of(parts) thereof.

The C-terminal fusion partner in the fusion lipoprotein in principle canbe any amino acid sequence comprising the at least one epitope.Preferably, the C-terminal fusion partner comprises an amino acidssequence of at least 5, 10, 15, 30, 50, 100, 150, 200, 250, 300, 350 or400 amino acids and/or no more than 800, 700, 600, 500 or 450 aminoacids. Preferably, the C-terminal fusion partner in the fusionlipoprotein of the invention is compatible with surface expression ofthe fusion lipoprotein when expressed in the Gram-negative bacterium.The compatibility of the C-terminal fusion partner with surfaceexpression of the fusion lipoprotein can be assayed as described above.

In one embodiment, the C-terminal fusion partner in the fusionlipoprotein of the invention is heterologous to the N-terminal fusionpartner. A C-terminal fusion partner that is heterologous to theN-terminal fusion partner is understood to mean that the amino acidsequences in the C-terminal fusion partner originate from one or moreprotein that are different than the protein from which the amino acidsequences in the N-terminal fusion partner originate. The heterologousC-terminal fusion partner can originate from the same organism as theN-terminal fusion partner, or the N-and C-terminal fusion partners canbe each from a different organism. Preferably, in a fusion lipoproteinaccording to the invention, the C-terminal fusion partner lacks aminoacid sequences from the surface exposed lipoprotein from which thesequences of the N-terminal fusion partner are derived. More preferably,the C-terminal fusion partner lacks a (contiguous) amino acid sequenceof at least 3, 4, 5, 6, 7, 8, 9, 10, 15 or at least 20 amino acids fromthe surface exposed lipoprotein from which the sequences of theN-terminal fusion partner are derived. Thus, preferably the C-terminalfusion partner comprises or consists of amino acids sequencesoriginating from one or more proteinaceous antigens that are differentfrom the surface exposed lipoprotein from which the amino sequences ofN-terminal fusion partner originate.

The C-terminal fusion partner in the fusion lipoprotein of theinvention, preferably comprises at least one epitope for inducing and/orenhancing an immune response against an antigen comprising the epitope.Preferably, a B-cell, humoral or antibody response is elicited by theepitope in the C-terminal fusion partner. Preferably the epitope in theC-terminal fusion partner elicits a protective and/or neutralizingantibody response. Alternatively and/or additionally, the C-terminalfusion partner comprises epitopes that elicit a T cell response. Apreferred T-cell response induced and/or enhanced by an immunogenicpeptide comprises at least one of an HLA class I restricted CTL responseand an HLA class II restricted Th response. More preferably the T-cellresponse consists of both an HLA class I restricted CTL response andsimultaneously an HLA class II restricted Th response, and may beadvantageously accompanied by a B-cell response.

The C-terminal fusion partner in the fusion lipoprotein can comprise oneor more epitopes from a wide range of antigens of pathogens (infectiousagents) and/or tumours. For example, the C-terminal fusion partner maycomprise one or more epitopes from antigens from pathogens andinfectious agents such as viruses, bacteria, fungi and protozoa. Someexamples of pathogenic viruses causing infections or tumours from whichepitopes from antigens may be derived include: hepatitis (A, B, or C),herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barrvirus), adenovirus, SV40 virus (causing mesothelioma), influenza virus,flaviviruses, ebola virus, echovirus, rhinovirus, coxsackie virus,coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus,measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus,dengue virus, molluscum virus, poliovirus, rabies virus, JC virus,arboviral encephalitis virus, and human immunodeficiency virus (HIVvirus; e.g., type I and II), human papilloma virus (HPV). Some examplesof pathogenic bacteria causing infections from which epitopes fromantigens may be derived include: Borrelia, Listeria, Escherichia,Chlamydia, Coxiella, Rickettsial bacteria, Mycobacteria, Staphylococci,Streptocci, Pneumonococci, Meningococci, Gonococci, Klebsiella, Proteus,Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli,Bordetella, bacteria causing Cholera, Tetanus, Botulism, Anthrax,Plague, Leptospirosis, Whooping cough and Lymes disease. Some examplesof pathogenic fungi causing infections from which epitopes from antigensmay be derived include: Candida (e.g., albicans, krusei, glabrata,tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus,niger), fungi of the genus Mucorales (Mucor, Absidia, Rhizopus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioidesbrasiliensis, Coccidioides immitis and Histoplasma capsulatum. Someexamples of pathogenic parasites causing infections from which epitopesfrom antigens may be derived include: Entamoeba histolytica, Balantidiumcoli, Naegleria, Fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii and Plasmodium falciparis.

In addition, the C-terminal fusion can comprise one or more epitopesfrom a wide range of tumour antigens, including e.g. MAGE, BAGE, RAGE,GAGE, SSX-2, NY-ESO-1, CT-antigen, CEA, PSA, p53, XAGE and PRAME butalso virally induced malignancies, comprising Human papilloma virus(HPV), Kaposi sarcoma herpes virus (KSHV), Epstein Bar virus inducedlymphoma's (EBV). Other examples of tumour antigens from which epitopesfor use in the present invention may be derived are various ubiquitouslyexpressed self-antigens that are known to be associated with cancer,which include e.g. p53, MDM-2, HDM2 and other proteins playing a role inp53 pathway, molecules such as surviving, telomerase, cytochrome P450isoform 1B1, Her-2/neu, and CD19 and all so-called house hold proteins.Cancers that may be treated in accordance with the present invention areselected among the following list: lung, colon, esophagus, ovary,pancreas, skin, gastric, head and neck, bladder, sarcoma, prostate,hepatocellular, brain, adrenal, breast, endometrial, mesothelioma,renal, thyroid, hematological, carcinoid, melanoma, parathyroid, cervix,neuroblastoma, Wilms, testes, pituitary and pheochromocytoma cancers.

In one embodiment, the C-terminal fusion partner comprises or consistsof one or more surface exposed epitopes from a proteinaceous antigen ofan infectious agent or tumour. The C-terminal fusion partner can e.g.comprises or consists of an extracellular and/or surface exposed domainof the proteinaceous antigen of an infectious agent or tumour.

In a preferred embodiment, the C-terminal fusion partner comprises orconsists of a surface exposed domain of a surface exposed bacterialprotein or lipoprotein. Preferably the surface exposed domain of asurface exposed protein or lipoprotein from a bacterium selected from agenus consisting of Bordetella, Borrelia, Coxiella Neisseria and any ofthe other pathogenic bacterial genera mentioned above. More preferablythe surface exposed domain is of a surface exposed Borrelia protein orlipoprotein selected from the group consisting of OspA, OspB, OspC,OspF, VlsE, BbCRASP1, Vsp 1, P35 (BBK32), P37 (BBK50), P39, P66, DpbAand BB017, as described in one or more of Schuijt et al. (2011, Trendsin parasitology. 27(1):40-7), Steere and Livey (2013; 49 in thereference list), Embers and Narasimhan (2013, Frontiers in cellular andinfection microbiology. 3:6) and Small et al. (2014, PloS one.9(2):e88245). Most preferably, the surface exposed domain comprises orconsists of amino acids 29-273 of SEQ ID NO: 4, amino acids 29-273 ofSEQ ID NO: 58, or amino acids 136-210 of SEQ ID NO: 59.

The amino acid sequence of the C-terminal fusion partner may also have35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity with the sequence of amino acids29-273 of SEQ ID NO: 4, amino acids 29-273 of SEQ ID NO: 58 or withamino acids 136-210 of SEQ ID NO: 59.

In a second aspect, the invention pertains to an OMV comprising a fusionlipoprotein as herein defined above. OMV (also known as “blebs”) for usein vaccines have traditionally been prepared by detergent extraction (adOMV purification process), wherein detergents such deoxycholate areused to remove LPS and increase vesicle release. The LPS of mostGram-negative bacteria, such as N. meningitidis is highly toxic, yetresidual amounts (approx. 1%) are needed in OMV to maintain vesiclestructure and for adjuvant activity. However, along with most of theLPS, the detergent extraction process also removes lipoproteins and istherefore not suitable for producing OMV comprising fusion lipoproteinsof the present invention. An OMV comprising a fusion lipoproteinaccording to the invention therefore preferably is not adetergent-extracted OMV. It is understood however, that a process forpreparing an OMV that is not a detergent-extracted OMV does not excludethe use of any detergents. The use of low concentration of detergentand/or the use of mild detergents are not excluded as long as most ofthe fusion lipoprotein according to the invention, i.e. at least 50, 60,70, 80, 90, 95 or 99% of the fusion lipoprotein, is maintained, e.g. ascompared the amount of fusion lipoprotein present in spontaneous orsupernatant OMV from an equal amount of the same culture.

A preferred OMV comprising a fusion lipoprotein of the invention is asupernatant or spontaneous OMV, i.e. sOMV as herein defined above, or anative OMV, i.e. nOMV as herein defined above. nOMV can be prepared asdescribed in Example 1.6 herein. Further methods for preparing nOMV aree.g. described in Saunders et al. (1999, Infect Immun, 67, 113-119), vande Waterbeemd et al. (2012, Vaccine, 30: 3683-3690) and in WO2013006055and methods for preparing sOMV are e.g. described in van de Waterbeemdet al. (2013, PLoS ONE, 8(1): e54314. doi:10.1371/journal.pone.0054314)and in Lee et al. (2007, Proteomics, 7: 3143-3153), all of which areincorporated herein by reference.

The OMV comprising a fusion lipoprotein of the invention are preferablyobtained/obtainable from a Gram-negative bacterium that has a geneticmodification selected from the group consisting of: (i) a geneticmodification that alters the lipopolysaccharide (LPS) biosynthesispathway, preferably in order to obtain less endotoxic and reactogenicvariants; (ii) a genetic modification that causes outer membraneretention of normally secreted antigens; (iii) a genetic modificationthat increases OMV production by removing outer membrane anchorproteins; (iv) a genetic modification that removes immune-modulatingcomponents which may trigger an undesired type of immune response; and,(v) a genetic modification that introduces expression of heterologousantigens from other pathogens than the host OMV producing strain.

The OMV comprising a fusion lipoprotein of the invention are preferablyobtained/obtainable from a Gram-negative bacterium that has a geneticmodification causing the bacterium to produce an LPS with reducedtoxicity but which LPS retains at least part of its adjuvant activity,wherein preferably the genetic modification reduces or eliminatesexpression of at least one of a lpxL1, lpxL2 and lpxK gene or ahomologue thereof and/or increases the expression of at least one of alpxE, lpxF and/or pagL genes. More preferably, the Gram-negativebacterium has a genetic modification reduces or eliminates expression ofan lpxL1 gene or a homologue thereof having at least 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity with the amino acid sequence of SEQ ID NO: 6.

The Gram-negative bacterium from which the OMV comprising a fusionlipoprotein of the invention are obtained/obtainable, further preferablycomprises a genetic modification that reduces or eliminates expressionof a gene encoding an anchor protein between outer membrane andpeptidoglycan in order to increase vesicle formation and therebyincrease OMV yield. A suitable genetic modification for this purposee.g. reduces or eliminates expression of an OmpA homologue, which arecommonly found in Gram-negative bacteria, e.g. the RmpM protein inNeisseria (Steeghs et al., 2002 Cell Microbiol, 4:599-611; van deWaterbeemd et al., 2010 Vaccine, 28:4810-4816). Thus, preferably, theGram-negative bacterium has a genetic modification reduces or eliminatesexpression of an rmpM gene or a homologue thereof having at least 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:7.

In one embodiment, the OMV comprising a fusion lipoprotein of theinvention are preferably obtained/obtainable from a Gram-negative thathas a genetic modification that reduces or eliminates expression of analP gene or a homologue thereof. The NalP protease has been identifiedas responsible for proteolytic release of the LbpB cell surface-exposedlipoprotein in Neisseria (Roussel-Jazédé et al., 2010, Infect Immun 78:3083-3089). In order to prevent proteolytic release of fusionlipoprotein of the invention, preferably, the Gram-negative host forproducing the OMV comprising a fusion lipoprotein of the invention has agenetic modification that reduces or eliminates expression of a nalPgene or a homologue thereof More preferably, expression of a nalP geneor homologue thereof is reduced or eliminated in a Gram-negative hostfor producing the OMV comprising a fusion lipoprotein wherein theN-terminal fusion partner comprises LbpB amino acid sequences.Preferably therefore, the Gram-negative bacterium has a geneticmodification reduces or eliminates expression of an nalP gene or ahomologue thereof having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identitywith the amino acid sequence of SEQ ID NO: 8.

A Gram-negative bacterial host cell for producing the OMV comprising afusion lipoprotein of the invention can further have one or more geneticmodifications that reduce or eliminate the expression of a gene selectedfrom the group consisting of cps, ctrA, ctrB, ctrC, ctrD, exbB, exbD,frpB, galE, htrB, msbB, lpbB, lpxK, lpxL1, nmb0033, opA, opC, rmpM,phoP, pilC, pmrE, pmrF, porA, porB, siaA, siaB, siaC, said, synA, synB,sync, tbpA and tbpB, or homologues thereof; many of these mutations arereviewed in WO02/09746.

A Gram-negative bacterial host cell for producing the OMV comprising afusion lipoprotein of the invention, preferably is bacterial host cellthat belongs to a genus selected from the group consisting of Neisseria,Bordetella, Escherichia and Salmonella, more preferably is bacterialhost cell that belongs to a species selected from the group consistingof Neisseria meningitidis, Bordetella pertussis, Escherichia coli andSalmonella enterica.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising an OMV as defined herein above, comprising afusion lipoprotein according to the invention. The composition furtherpreferably comprises a pharmaceutically acceptable carrier, medium ordelivery vehicle as are conventionally known in the art (see e.g.“Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7^(th) edition,2012, www.pharmpress.com). Pharmaceutically acceptable stabilizingagents, osmotic agents, buffering agents, dispersing agents, and thelike may also be incorporated into the pharmaceutical compositions. Thepreferred form depends on the intended mode of administration andtherapeutic application. The pharmaceutical carrier can be anycompatible, non-toxic substance suitable to deliver the activeingredients, i.e. the OMV comprising the fusion protein of the inventionto the patient.

Pharmaceutically acceptable carriers for parenteral delivery areexemplified by sterile buffered 0.9% NaCl or 5% glucose optionallysupplemented with a 20% albumin. Alternatively, the OMV comprising thefusion protein can be suspended in Phosphate buffer saline (PBS).Preparations for parental administration must be sterile. The parentalroute for administration of the OMV comprising the fusion protein of theinvention is in accord with known methods, e.g. injection or infusion byintravenous, intraperitoneal, intramuscular, intraarterial orintralesional routes. A typical pharmaceutical composition forintramuscular injection would be made up to contain, for example, 1-10ml of phosphate buffered saline comprising the effective dosages of theOMV comprising the fusion protein of the invention. Methods forpreparing parenterally administrable compositions are well known in theart and described in more detail in various sources, including, forexample, “Remington: The Science and Practice of Pharmacy” (Ed. Allen,L. V. 22nd edition, 2012, www.pharmpress.com).

In another aspect, the invention pertains to an OMV comprising a fusionprotein of the invention or a pharmaceutical composition comprising saidOMV for use as a medicament.

In another aspect, the invention pertains to an OMV comprising a fusionprotein of the invention or a pharmaceutical composition comprising saidOMV for the prevention or treatment of an infectious disease or tumourassociated with an antigen as herein defined above. Preferably, theinfectious disease is a Borrelia infection, more preferably a Borreliaburgdorferi infection.

In this aspect, the invention thus relates to a method for vaccinationagainst, or for prophylaxis or therapy of an infectious disease ortumour by administration of a therapeutically or prophylacticallyeffective amount of (a pharmaceutical composition comprising) an OMVcomprising a fusion protein of the invention, to a subject in need ofprophylaxis or therapy. The invention also relates to an OMV comprisinga fusion protein of the invention for use as a medicament, preferably amedicament for vaccination against, or for prophylaxis or therapy of aninfectious disease or tumour.

In yet another aspect, the invention relates to a nucleic acid moleculeencoding a pre-profusion lipoprotein, wherein upon expression in aGram-negative bacterium the pre-profusion lipoprotein matures into thefusion lipoprotein as defined herein above, and wherein preferably thenucleic acid molecule is an expression construct for expression of thepre-profusion lipoprotein in a Gram-negative bacterium. Means andmethods for constructing expression constructs for expression of theprotein Gram-negative bacteria are generally well-known in the art.

In again a further aspect, the invention relates to a Gram-negative hostcell comprising a nucleic acid molecule or an expression construct asdefined above. Preferably the host cell is bacterial host cell thatbelongs to a genus or species as defined above.

In a final aspect, the invention relates to a method for producing anOMV comprising a fusion lipoprotein of the invention. The methodpreferably comprises the steps of: a) cultivating a Gram-negative hostcell comprising a nucleic acid molecule or an expression construct asdefined above for expression in the host cell of a pre-profusionlipoprotein, wherein upon expression in a Gram-negative host cell thepre-profusion lipoprotein matures into the fusion lipoprotein as definedherein above; and, c) recovering the OMV, wherein the recovery at leastcomprises removal of the bacteria from the OMV. Preferably in themethod, the recovery of the OMV in step c) is preceded by a step b),wherein the OMV are extracted. The method for producing OMV comprising afusion lipoprotein of the invention is further preferably, adetergent-free method as herein defined and described above.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

DESCRIPTION OF THE FIGURES

FIG. 1. Schematic overview of fHbp (hatched), OspA (white) and fusionconstructs. Amino acid numbering for fHbp and OspA is shown in boxedareas. S.p.=signal peptide, α1=N-terminal alpha-helix of fHbp includingthe subsequent loop, β1-4=first four N-terminal beta-sheets of fHbp.Numbered arrows refer to primers listed in Table 1, forward primers areshown above and reverse primers are shown below the schematic (fusion)genes, positioning of the arrow heads reflects the approximatepositioning of the 3′ end. The artificial linker (6) that wasincorporated in some constructs is shown in white. Note that constructfA1 was used as template to create constructs fA3b and fA5c.

FIG. 2. Western blot analysis of OspA expression in N. meningitidiscells (A) or nOMVs (B) carrying OspA or fusion constructs. Cells andnOMVs were normalized based on OD₆₀₀ and protein content, respectively.Expected molecular mass of the different constructs is shown in kDa (inparentheses). Arrows indicate the full-length proteins. Wt: N.meningitidis cells (or nOMVs harvested from these cells) carrying‘empty’ plasmid pEN11-Imp (see Materials and Methods).

FIG. 3. Western blot analysis of mouse sera. Lanes were loaded with E.coli carrying pEN11-OspA (‘OspA’) or pEN11-Imp (‘Imp’). (A) From left toright; (1) control blot with anti-OspA showing the expected band of ˜28kDa and blots with pooled sera of groups of five mice immunized with (2)PBS, (3) high dose empty nOMVS, (4) high dose OspA nOMVs, (5) high dosefA1 nOMVs, (6) low and (7) high dose fA4b nOMVs, and (8) low and (9)high dose of fA6 nOMVS. (B) Blots with sera from individual miceimmunized with a high dose of fA6 nOMVs (pooled sera of this group areat the far right in FIG. 4A). Arrows point at bands with a molecularweight similar to that of OspA (˜28 kDa).

FIG. 4. ELISA data for sera from individual mice immunized with 20 μg/mlnOMVs carrying construct fA4b, fA4c, fA6, or fA7. Pooled sera of miceimmunized with 20 μg/ml nOMVs carrying empty vector pEN11-Imp were usedas negative control and subtracted from data before plotting. Note thatsera of mice immunized with fA6 tested here are the same as the ones inthe Western blot shown in FIG. 4b , which is reflected in the lowerresponsiveness of individual #2.

FIG. 5. Schematic overview of TbpB (hatched) and OspA (white) fusionconstructs. Amino acid numbering for TbpB and OspA is shown in boxedareas. S.p.=signal peptide. Numbered arrows refer to primers listed inTable 1, forward primers are shown above and reverse primers are shownbelow the schematic (fusion) genes, positioning of the arrow headsreflects the approximate positioning of the 3′ end.

FIG. 6. Western blot analysis of OspA expression in N. meningitidiscells carrying constructs fTA1-4 using OspA polyclonal (RocklandImmunochemicals). Expected molecular mass of the different constructs isshown in kDa (in parentheses). Arrows indicate full-length proteins.

FIG. 7. Schematic overview and western blot analysis of fHbp-OspA andTbpB-OspA fusion constructs. A) Schematic overview of the constructs.fHbp fragments are hatched, OspA fragments are grey and TbpB fragmentsare white. Amino acid numbering for fHbp, TbpB and OspA is shown inboxed areas. S.p.=signal peptide. Numbered arrows refer to primerslisted in Table 1, forward primers are shown above and reverse primersare shown below the schematic (fusion) genes, positioning of the arrowheads reflects the approximate positioning of the 3′ end. B) Westernblot analysis of OspA expression in N. meningitidis cells carryingconstructs fA10 and fTA5 using OspA monoclonal antibody (Santa CruzBiotechnologies). Expected molecular mass of the different constructs isshown in kDa (in parentheses). Arrows indicate full-length proteins.

FIG. 8. Schematic overview and western blot analysis of fHbp-OspC fusionconstructs. A) Schematic overview of the constructs. fHbp fragments arehatched and OspC fragments are white. Amino acid numbering for fHbp andOspC is shown in boxed areas. S.p.=signal peptide. Numbered arrows referto primers listed in Table 1, forward primers are shown above andreverse primers are shown below the schematic (fusion) genes,positioning of the arrow heads reflects the approximate positioning ofthe 3′end. B) Western blot analysis of OspC expression in N.meningitidis cells with constructs fC9 and OspC. No clear expression forOspC was observed. Expected molecular mass of the different constructsis shown in kDa (in parentheses). The construct fC9 does not formhomodimers.

FIG. 9. Schematic overview and western blot analysis of the fHbp-RmpMand TbpB-RmpM fusion constructs. A) Schematic overview of theconstructs. fHbp fragments are hatched, RmpM fragments are grey and TbpBfragments are white. Amino acid numbering for fHbp, TbpB and RmpM isshown in boxed areas. S.p.=signal peptide.

Numbered arrows refer to primers listed in Table 1, forward primers areshown above and reverse primers are shown below the schematic (fusion)genes, positioning of the arrow heads reflects the approximatepositioning of the 3′ end. B) Western blot analysis of RmpM expressionin N. meningitidis ARmpM cells with constructs fR1 and fTR1. Expectedmolecular mass of the different constructs is shown in kDa (inparentheses). Arrows indicate full-length proteins.

EXAMPLES 1. Materials and Methods 1.1 Antibiotics

Ampicillin (Amp) and chloramphenicol (Cam) were purchased from Sigma.Stock solution were prepared in Milli-Q (MQ) water, filter-sterilizedusing a 0.22 μm Steriflip (Milipore), and stored at 4° C.

1.2 Bacterial Strains and Growth Conditions

Escherichia coli strains JM109 (Promega) and TOP1OF′ (Invitrogen) wereused for cloning steps involving vectors pGEM-T Easy and pEN11,respectively. Both strains were grown at 37° C. on Luria Bertani medium(MP Biomedicals) supplemented with 15 gram agar/liter and appropriateantibiotics (50 μg/ml Amp for pGEM-T Easy and 25 μg/ml Cam for pEN11).For blue/white screening of JM109, plates were supplemented with 50μg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal,Fermentas) and 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG,Thermo Scientific). Liquid cultures were grown at 37° C. and 200 RPM.

Neisseria meningitidis strain HB-1 carrying the lpxL1 deletion (52) wasgrown on Difco GC medium base supplemented with IsoVitaleX (both BectonDickinson) at 37° C. in a humid atmosphere containing 5% CO₂. Plateswere supplemented with 3 μg/ ml Cam in case of transformation withpEN11. Liquid cultures were grown in Tryptic Soy Broth (TSB, BectonDickinson) with 3 μg/ml Cam and 1 mM IPTG at 37° C. and 150 RPM.

Borrelia burgdorferi strain B31 was kindly supplied by the lab of J.Hovius (Amsterdam Medical Centre). Genomic DNA of B. burgdorferi wasextracted using the DNeasy Blood & Tissue Kit (Qiagen) according to themanufacturer's instructions.

1.3 Recombinant DNA Technology

All primers used in this study are shown in Table 1. Hybrids were madeof fHbp (N. meningitidis), TbpB (N. meningitidis), OspA (B. burgdorferior B. afzelii), OspC (B. burgdorferi) and RmpM (N. meningitidis). Allhybrids were constructed using Overlap Extension PCR (53). All PCRs werecarried out using the Accuprime Taq DNA Polymerase System (Invitrogen)to ensure both high fidelity amplification and the addition of 3′A-overhangs. See FIGS. 1 and 5 for a schematic overview of the differentconstructs.

TABLE 1 Oligonucleotide primers used in this study SEQ Primer ID Primernumber NO name Sequence (5′ → 3′)  1  9 fHbp-F CATATGCGCCGTTCGGACGACATTTGATTTTTGC  2 10 fHbp-R GACGTCACGGTAAATTATCGT GTTCG  3 11 OspA-FCATATGAAGGAGAATATATTA TGAAAAAATATTTATTGG  4 12 OspA-RGACGTCTAAAGCTAACGCTAA AGCAAATCC  5 13 M13-F GTAAAACGACGGCCAG  6 14 M13-RCAGGAAACAGCTATGAC  7 15 pEN11-F AAACCGCATTCCGCACCACAA G  8 16 pEN11-RGGGCGACACGGAAATGTTGAA TAC  9 17 fA1-F GTGTCGCCGCCGACATCGGTGCGAGCGTTTCAGTAGATTTGC 10 18 fA1-R GCAAATCTACTGAAACGCTCGCACCGATGTCGGCGGCGACAC 11 19 fA2b-F GGCGACCCGGGTGGCTCAGGTGCTAGCGTTTCAGTAGATTTG C 12 20 fA2b-R AAACGCTAGCACCTGAGCCACCCGGGTCGCCGATTCTGAACT G 13 21 fA3b-F TTAAAACCGGGTGGCTCAGGTGCTAGGGCGACATATCGCGGG ACG 14 22 fA3b-R CGCCCTAGCACCTGAGCCACCCGGTTTTAAAGCGTTTTTAAT TTC 15 23 fA4b-F AAGCAACCGGGTGGCTCAGGTGCTAGCGTTTCAGTAGATTTG C 16 24 fA4b-R AAACGCTAGCACCTGAGCCACCCGGTTGCTTGGCGGCAAG 17 25 fA4c-F CCTTGCCGCCAAGCAATGTAA GCAAAATGTTAGC 1826 fA4c-R GCTAACATTTTGCTTACATTG CTTGGCGGCAAGG 19 27 fA5c-FGAAATTAAAAACGCTTTAAAA TGCAGCAGCGGAGGGGGTGGT G 20 28 fA5c-RCACCACCCCCTCCGCTGCTGC ATTTTAAAGCGTTTTTAATTT C 21 29 fA6-FCCATAAAGACAAAGGTTTGAG CGTTTCAGTAGATTTGC 22 30 fA6-RGCAAATCTACTGAAACGCTCA AACCTTTGTCTTTATGG 23 31 fA7-FCAATACGGGCAAATTGAAGAG CGTTTCAGTAGATTTGC 24 32 fA7-RGCAAATCTACTGAAACGCTCT TCAATTTGCCCGTATTG 25 33 pfHbpTbpB-FCGTATGACTAGGAGTAAACCT ATGAACAATCCATTGGTGAAT CAGG 26 34 pfHbpTbpB-RCCAATGGATTGTTCATAGGTT TACTCCTAGTCATACG 27 35 TbpB-RGACGTCCGTCTGAAGCCTTAT TCTCG 28 36 OspA afz-R GACGTCTACTTTTTGGCTCAG longTACC 29 37 OspC-F CATATGAATAAAAAGGAGGCA CAAATTAATG 30 38 OspC-RGACGTCTTAATTAAGGTTTTT TTGGACTTTCTG 31 39 RmpM-R GACGTCGCATCGGCAAGATATTGC 32 40 fA10-F CCATAAAGACAAAGGTTTGAG CGCTTCAGTAGATTTGC 33 41 fA10-RGCAAATCTACTGAAGCGCTCA AACCTTTGTCTTTATGG 34 42 fTA1-FCCTGTGTTTTTGTTGAGTGCT TGTAAGCAAAATGTTAGCAGC CTTG 35 43 fTA1-RCAAGGCTGCTAACATTTTGCT TACAAGCACTCAACAAAAACA CAGG 36 44 fTA2-FGCAAGCCCAAAAAGACCAAAG CGTTTCAGTAGATTTGC 37 45 fTA2-RGCAAATCTACTGAAACGCTTT GGTCTTTTTGGGCTTGC 38 46 fTA3-FCAAGCGGCGGAATTGGTATCA GAGGAGCGTTTCAGTAGATTT GC 39 47 fTA3-RGCAAATCTACTGAAACGCTCC TCTGATACCAATTCCGCCGCT TG 40 48 fTA4-FGGATGATGGTGATATCAAAAG CGTTTCAGTAGATTTGCCTGG TG 41 49 fTA4-RCACCAGGCAAATCTACTGAAA CGCTTTTGATATCACCATCAT CC 42 50 fTA5-FGCAAGCCCAAAAAGACCAAAG CGCTTCAGTAGATTTGC 43 51 fTA5-RGCAAATCTACTGAAGCGCTTT GGTCTTTTTGGGCTTGC 44 52 fC9-FGACCATAAAGACAAAGGTTTG AATAAATTAAAAGAAAAACAC ACAG 45 53 fC9-RCTGTGTGTTTTTCTTTTAATT TATTCAAACCTTTGTCTTTAT GGTC 46 54 fR1-FCCATAAAGACAAAGGTTTGCC GCAATATGTTGATGAAACC 47 55 fR1-RGGTTTCATCAACATATTGCGG CAAACCTTTGTCTTTATGG 48 56 fTR1-FGCAAGCCCAAAAAGACCAACC GCAATATGTTGATGAAACC 49 57 fTR1-RGGTTTCATCAACATATTGCGG TTGGTCTTTTTGGGCTTGC

Amplicons were ligated blunt-end in the pGEM-T Easy vector (Promega) andsubsequently heat-shock transformed into E. coli JM109 cells (Promega)according to the manufacturer's instructions. Transformants werescreened for insert of the correct length using primers M13-F and M13-R(see Table 1). Plasmids were isolated from overnight cultures ofpositive JM109 transformants using the Wizard Plus SV Plasmid MiniprepSystem (Promega). Isolated plasmids were digested using restrictionenzymes AatII and NdeI (Fermentas). The resulting fragments were thenseparated by gel-electrophoresis, and subsequently gel-purified usingthe Wizard SV Gel and PCR Cleanup System (Promega). Universal plasmidpEN11 (54) was used as vector after digestion with AatII and NdeI in thepresence of Shrimp Alkaline Phosphatase (Roche). Inserts and vector wereligated using T4 DNA ligase (Promega) according to the manufacturer'sinstruction and the resulting plasmids were then heat-shock transformedinto E. coli One Shot TOP10F′ competent cells (Invitrogen).Transformants were screened for insert of the correct length usingprimer pEN11-F and a reverse primer of the respective construct.Approximately 1 μg of isolated pEN11 plasmid (isolation procedure asdescribed before) carrying OspA or fHbp-OspA fusions was added to a 1 mlsuspension of N. meningitidis cells (OD₆₀₀≈0.2) and grown in TSBsupplemented with 10 mM MgCl₂ at 37° C. for 6 hours (no shaking).Bacteria were then plated on GC plates containing 3 μg/ml Cam. Colonieswere isolated after 24-48 hours and screened for the presence of pEN11with correct insert as before.

1.4 Expression of Constructs in N. meningitidis Cells and nOMVs

N. meningitidis cells were streaked out on GC II plates (BectonDickinson) and grown overnight as described. The following day, colonieswere harvested with a sterile cotton swab were suspended in TSBcontaining 1 mM IPTG and 3 μg/ml Cam and further diluted to an OD₆₀₀ of0.2 in 5 ml of the same broth with supplements. Cells were grown for 4hours at 37° C. and 170 RPM after which OD₆₀₀ was again determined andaliquots corresponding to 4.0×10⁸ CFU were centrifuged for 5 min at13,000 RPM and resuspended in phosphate buffered saline (PBS). Cellswere then centrifuged as before, after which the resulting pellet wasdissolved in 40 μl MQ, combined with 10 μl 5× sample buffer (50%glycerol, 0.25% Tris pH 6.8, 10% Sodium Dodecyl Sulphate, 10%dithiothreitol and 0.05% Bromophenol blue), and boiled for 10 minutes.Samples were then stored at −20° C. for further analysis.

N. meningitidis native OMVs (nOMVs) were diluted to 20 μg total proteinper ml in PBS, after which 40 μl nOMV suspension was boiled with 10 μ15×sample buffer as before.

Protein samples of cells of nOMVs were separated by SDS-PAGE on 12%Precise Protein Gels (Thermo Scientific). Separated proteins were thentransferred to 0.45 μm nitrocellulose membranes (BioRad). Membranes wereincubated for 1 hour on a rolling table in a 1:1000 dilution ofanti-OspA (Rockland) in buffer containing 0.1M Tris, 1.54 M NaCl, and 5%Tween-80. The membrane was then transferred to a 1:2000 dilution ofgoat-anti-rabbit IgG AP (Southern BioTech) in the same buffersupplemented with 0.5% Protifar (Nutricia). Blots were developed usingthe AP Conjugate Substrate Kit (BioRad).

1.5 Immunostaining

N. meningitidis cells carrying pEN11 with the various constructs wereimmobilized on coverslips coated with poly-1-lysine (Sigma). Cells werefixated with 2% formaldehyde in PBS for 10 minutes. After blocking inPBS containing 3% Bovine Serum Albumin (BSA, Sigma), the coverslips werefirst incubated in a 1:300 dilution of a mix of anti-OspA (Rockland) andanti-fHbp (variant 1, NIBSC) in PBS with 0.5% BSA. After washing, theywere incubated in a 1:300 dilution of a mix of Alexa Fluor 488goat-anti-rabbit IgG and Alexa Fluor 594 goat-anti-mouse IgG (LifeTechnologies). Slides were post-fixed in 2% formaldehyde in PBS andviewed under an Olympus CKX41 fluorescence microscope at 40xmagnification using appropriate filters.

1.6 Purification of nOMV Vaccines

Glycerol-stocks of clones selected for the immunization experiment werestreaked out on GC II plates and grown overnight under conditionsdescribed above. The following day, colonies were harvested and used tostart a 200 ml culture of OD₆₀₀=0.05 in TSB with 1 M IPTG and 3 μg/mlCam. These cultures were grown at 37° C. and 130 RPM and OD₆₀₀ wasmeasured at regular intervals. When cultures reached an OD₆₀₀ of 1.5(after ˜6 hours) they were placed on ice and subsequently centrifuged at3,500 RPM and 4° C. for 30 minutes. Pellets were resuspended in aTris-EDTA buffer (100 mM Tris, 10 mM EDTA, pH=8.6) and incubated on ahorizontal shaking table for 30 minutes. Since this buffer contains achelating agent (EDTA) that destabilizes the OM, the release of OMVs isstimulated. The suspensions were centrifuged at 13,000 RPM for 30minutes and the supernatant was sterilized using a Steriflip 0.22 μmfilter (Millipore). The sterile supernatant was then centrifuged at40,000 RPM for 65 minutes, after which the resulting OMV pellet wasallowed to dry before being resuspended in 1 ml sucrose buffer.Suspensions were again filtered as before and stored at 4° C.

The nOMV isolation procedure described above was developed in order toharvest as many nOMVs as possible without the hitchhiking of othercellular proteins due to lysis. As we noticed that the expression ofOspA, fA1, fA2b, fA3b, and fA5c in nOMVs harvested using this procedurecould be increased by allowing the respective cultures to grow for 12hours, without significantly increasing the hitchhiking of otherbacterial proteins (data not shown). We therefore decided to use thealternative isolation procedure for these constructs, in order toequalize the expression of constructs as much as possible.

The total protein concentration of the isolated nOMVs was measured usingthe BCA Protein Assay Kit (Pierce) and nOMVs were then further dilutedin PBS to 5 or 20 μg total protein per ml on the day of vaccination.

1.7 Mice and Immunization

Groups of five female, six- to eight-week-old BALB/cOlaHsd mice (Harlan)were immunized subcutaneously with 200 μl of nOMVs, at either lowconcentration (5 ng/ml) or high concentration (20 ng/ ml). Next to thegroups that received nOMVs ‘loaded’ with OspA (two groups) or fHbp-OspAfusions (sixteen groups), two control groups received ‘empty’ nOMVSharvested form cells carrying the pEN11 plasmid with the imp genereplacing the ospA-constructs (54). An additional control group wasimmunized with PBS, resulting in a total of 21 groups. Mice wereimmunized at days 0 and 28 and sacrificed 14 days after the lastimmunization. Blood was collected in Vacuette Z Serum Clot Activatortubes (Greiner Bio-One) and centrifuged at 2000 RPM for 15 minutes.Subsequently, sera were collected and stored at −20° C. for furtheranalysis.

1.8 Analysis of Sera

Sera were first pooled by group (five mice) and analyzed for thepresence of antibodies by Western blot. Membranes were loaded withproteins from E. coli TOP10F′ cells carrying either pEN11-Imp orpEN11-OspA. Membranes were incubated for one hour on a rolling tablewith pooled sera diluted 1:1000 in Tris buffer (described previously),followed by incubation in a 1:2000 dilution of secondary antibody(goat-anti-mouse IgG AP, Southern BioTech) in the same buffer and blotdevelopment as described previously. The ten individual sera of all micefrom the two most strongly reacting groups (fA4b, 20 μg/ml and fA6, 20μg/ml) were analyzed in the same manner.

For ELISAs, 100 μl of 0.5 μg/ml OspA control protein (Rockland) dilutedin PBS was coated on the surface of wells in Microlon 96-well plates(GreinerBio) and incubated overnight at room temperature (RT). Thefollowing day, plates were blocked by adding 200 μl 0.5% Protifar in PBSfollowed by incubation for 30 minutes at RT. Plates were then washedthree times in wash buffer (water with 0.05% tween-80). Sera ofindividual mice were suitably diluted in PBS with 0.1% tween-80 and 100μl was added per well, followed by incubated for 1 hour at RT. Plateswere then again washed three times in wash buffer, after which 100 μl ofgoat-anti-mouse IgG HRP (SouthernBiotech) was added (diluted 1:4000 inPBS with 0.1% tween-80). After incubation for 1 hour, the plates werewashed as before and 100 μl of TMB was added. Plates were then incubatedfor 10 minutes after which coloring was stopped with 100 μl 2M H₂SO₄.OD₄₅₀ was subsequently measured on a SynergyMx plate-reader (Biotek).

2. Results

2.1 Construction of fHbp-OspA Fusion-Genes

Adjacent to their lipidated N-terminal cysteine, most characterizedlipoproteins contain a stretch of amino acids with a low propensity forthe formation of secondary structure. This so-called ‘tether’ is thoughtto act as a flexible linker between the lipid ‘anchor’ (the lipidatedcysteine) and the structurally confined part of the protein (55).Tethers also play a role in the transport of lipoproteins over the outermembrane, since deletions in this region can result in mislocalizationof surface-exposed lipoproteins to the inside of the outer membrane(55).

Since we found no evidence for the surface exposure of OspA expressed inNeisseria, we hypothesized that the switch of bacterial host resulted inmislocalization of the protein to the inside of the outer membrane. Wethen set out to test whether the addition of parts of fHbp, asurface-exposed Neisserial lipoprotein, might correct thismislocalization. Since the sorting rules for transport over the outermembrane in Neisseria have not yet been elucidated, we designed hybridgenes that combined various parts of fHbp with the globular domain ofOspA.

A schematic overview of fHbp and OspA, as well as the fusion genescreated from these two genes by overlap extension PCR, is given inFIG. 1. Information regarding the structure of both lipoproteins wasobtained from published crystal structures (48, 56). Both fHbp and OspAcontain a signal peptide (that is cleaved after transport over the innermembrane) and a tether region. The globular domain of fHbp consists ofan N-terminal and a C-terminal domain that are separated by a 15 aminoacid linker.

Genomic DNA of N. meningitidis strain 44/76 was used as template in allPCR reactions involving the amplification of parts of fHbp. The fHbp-Fprimer anneals upstream of the fHbp promoter (57), and therefore allfusion-genes contain the fHbp promoter. OspA was amplified from genomicDNA of B. burgdorferi strain B31 using primers OspA-F and OspA-R (Table1), and was successfully expressed in E. coli TOP10F′ from the pEN11plasmid, which contains an IPTG-inducible tac-lacUV5 promoter (54).

The six amino acid linker peptide that was introduced in constructsfA2b, fA3b, and fA4b was previously used to successfully link OspA tocalmodulin (58). We created eight different fusion constructs betweenfHbp and OspA. From here on we refer to these constructs as ‘fA’.Primers used for the construction of fusion genes are shown above(forward primers) or below (reverse primers) the schematic genes in FIG.1 (primer numbers refer to Table 1). All fusion genes were created byoverlap extension PCR, a two-step PCR protocol (53). In short, the geneparts to be fused were first amplified separately using partiallyoverlapping primers, after which both parts (that can anneal to eachother) were used as template in a second reaction yielding the fusiongene. As an example, the first step PCRs for construct fA1 used primerpairs 1-10 (to amplify the fHbp promoter, signal peptide, and tetherfrom the N. meningitidis genome) and 4-9 (to amplify the globular domainof OspA from the B. burgdorferi genome). The resulting PCR products werethen mixed and used as template in a second PCR reaction. In this secondreaction, both PCR products anneal to each other and at the same timeserve as template for primer pair 1-4 resulting in the full-length PCRproduct fA1. All other fusion genes were created using the same method.Note that construct fA1 served as template for the construction of fA3band fA5c.

In fA1, the signal peptide and tether of fHbp were fused to the globulardomain of OspA. In constructs fA2b and fA3b, the C-terminal domain(fA2b) or N-terminal domain (fA3b) of fHbp was replaced by the globulardomain of OspA and connected via an artificial linker (PGGSGA). Inconstructs fA4b and fA4c, the complete fHbp gene was linked to theglobular domain of OspA using the artificial linker (fA4b) or the OspAtether (fA4c). Construct fA5c is fA1 linked to the globular domain offHbp via the fHbp tether. Constructs fA6 is similar to fA1, but inaddition to signal peptide and tether also contains the firstalpha-helix and subsequent loop of the N-terminal domain of fHbp.Construct fA7 is similar to fA6, but additionally contains the firstfour beta-sheets of the N-terminal domain of fHbp. All constructs weresuccessfully expressed from pEN11 in E. coli TOP10F′ beforetransformation in Neisseria (data not shown).

2.2 Construction of TbpB-OspA Fusion Genes

TbpB of N. meningitidis H44/76 contains a signal peptide (residues1-20), a 38 amino acid flexible linker or ‘tether’ (residues 21-58), anda large globular domain (residues 59-691). In order to test whetherfusion of N-terminal parts of TbpB to OspA could rescue surfaceexpression of OspA in N. meningitidis, four TbpB-OspA fusion constructswere designed. Because TbpB has an iron-regulated promoter, the TbpBpromoter was first exchanged for the constitutive fHbp promoter usingoverlap extension PCR with primers 1, 25-27 (see Table 1). The resultingconstruct ('TbpB with fHbp promoter', see FIG. 5) was then used astemplate for fusion PCRs between TbpB and OspA.

An overview of constructs fTA1-4 can be found in FIG. 5, while primersfor the overlap-extension PCRs can be found in Table 1. Genomic DNA fromNeisseria meningitidis H44/76 was used as template for amplification ofparts of TbpB and genomic DNA of Borrelia burgdorferi strain B31 wasused as template for the OspA part.

Construct fTA1 contained residues 1-20 (the signal peptide) of TbpBfused to residues 17-273 (tether and globular domain) of OspA. ConstructfTA2 contained residues 1-58 (signal peptide and tether) of TbpB fusedto residues 29-273 (globular domain) of OspA. Construct fTA3 containedresidues 1-75 (signal peptide, tether and first seventeen N-terminalamino acids of the globular domain) of TbpB fused to residues 29-273(globular domain) of OspA. Construct fTA4 contained residues 1-99(signal peptide, tether and first 41 N-terminal amino acids of theglobular domain) of TbpB fused to residues 29-273 (globular domain) ofOspA. TbpB fusion points for fTA3 and fTA4 were chosen in loops betweensecondary structure elements, based on the TbpB crystal structure (63).

To test whether OspA serotypes other than serotype 1 (B. burgdorferiB31) could be transported to the meningococcal cell surface with thehelp of N-terminal parts of fHbp or TbpB, fusion constructs similar tofA6 and fTA2 were made, the only difference being that they containedthe globular domain of OspA serotype 2 from Borrelia afzelii PKo(residues 29-273). All constructs were made by overlap extension PCR,see FIG. 7 and Table 1 for details.

2.3 Construction of fHbp-OspC Fusion Genes

OspC is a surface-exposed borrelial lipoprotein. It is generallyconsidered to be the most interesting vaccinogen after OspA (64).However, the expression of full-length OspC including its signalsequence has proven difficult in E. coli (65), possibly due to itstendency to aggregate; in the Borrelia outer membrane, it is present inthe form of large multimeric complexes. Expression of full-length OspCin both E. coli and N. meningitidis previously led to similar problemsin our hands, resulting in poor expression on Western blots (data notshown).

In Borrelia, OspC forms homo-dimers on the cell-surface, mostly byinteractions between the N-terminal α1-helices (66, 67). Since mostmurine and human OspC epitopes are located on the C-terminal side of theprotein (68, 69), fusion construct fC9 (FIG. 8) was generated, thatcombined the previously described N-terminal part of fHbp (as used infA6) and C-terminal residues 136-210 of OspC (see FIG. 8). To this end,genomic DNA of N. meningitidis strain H44/76 was used as template forthe amplification of fHbp and genomic DNA of B. burgdorferi strain B31was used for amplification of OspC.

2.4 Construction of fHbp-RmpM and TbpB-RmpM Fusion Genes

RmpM is an outer membrane protein of N. meningitidis that is thought toassociate non-covalently with the peptidoglycan layer (70). It consistsof a signal peptide, a flexible N-terminal domain (which binds tointegral outer membrane proteins), and an OmpA-like C-terminal domain(which is thought to associate with peptidoglycan). Since RmpM isgenerally considered to be mostly periplasmic, an attempt was made toexpress RmpM at the cell surface of N. meningitidis. It was decided toleave out the first 89 residues that consist of signal peptide and theunstructured N-terminal domain. The remaining C-terminal domain(residues 90-242) was fused to N-terminal parts of fHbp (residues 1-50)and TbpB (residues 1-58) that had been used previously for thesuccessful surface localization of OspA. Genomic DNA of N. meningitidisstrain H44/76 was used as template for the amplification of fHbp, TbpB,and RmpM. The constructs were named fR1 and fTR1 (see FIG. 9).

2.5 OspA and fHbp-OspA Fusions are Expressed in N. meningitidis Cellsand nOMVs

The expression of the different constructs in N. meningitidis is shownin FIG. 2A and expression in nOMVs harvested from these cells is shownin FIG. 2B. Expression levels in N. meningitidis cells clearly varybetween the different constructs and similar variation is observed inthe nOMVs, with high expression levels for constructs fA4b, fA4c, fA6,and fA7. Several constructs are apparently prone to degradation. Forsome constructs (fA3b, fA5c), degradation seems to be amplified in nOMVscompared to cells.

2.6 At Least Four fHbp-OspA Hybrids are Surface Exposed

We tested whether or not OspA and the eight fHbp-OspA fusions weresurface-localized in N. meningitidis using immunostaining. Briefly,cells containing plasmid pEN11 with the various constructs wereincubated with a mix of anti-OspA and anti-fHbp (positive control),followed by incubation with fluorescent secondary antibodies (green forOspA and red for fHbp). Cells containing constructs fA4b, fA4c, fA6, andfA7 showed clear green fluorescence, indicating surface exposure ofthese constructs (data not shown). No green fluorescence was observedfor OspA or any of the other constructs (data not shown), indicatingthat they were not surface exposed, although we cannot rule out thepossibility that this is due to their lower expression level (see FIG.2A).

2.7 Expression and Surface-Exposure TbpB-OspA Hybrids

Successful expression of all four constructs in E. coli and N.meningitidis was confirmed by Western blot using a polyclonal antiserumagainst OspA (Rockland Immunochemicals—see FIG. 6 for Neisseria Westernblots). Immunostaining (as described above) showed no signal forconstruct fTA1, but a strong signal for constructs fTA2, fTA3, and fTA4(data not shown). This proves that N-terminal parts of TbpB can be usedfor the surface localization of OspA and that at least signal peptideand tether are required for this.

2.8 Expression and Surface-Exposure of Alternative C-Protein FusionPartners 2.8.1 Expression and Surface Exposure of an Alternative OspASerotype

Successful expression of both constructs in E. coli and N. meningitidiswas confirmed by Western blot using a polyclonal antiserum against OspA(Rockland Immunochemicals—see FIG. 7 for Neisseria Western blots).Immunostaining with the OspA polyclonal (Rockland Immunochemicals)proved difficult, most probably because the antiserum (which was raisedagainst serotype 1 OspA from B. burgdorferi) showed considerably weakerbinding to the B. afzelii OspA. Therefore, an OspA monoclonal antibody(Santa Cruz Biotechnology) was used for immunostaining. N. meningitidiscells expressing constructs fA10 and fTA5 showed a strong signal whenthis monoclonal was used for immunostaining (data not shown), indicatingthat the serotype 2 OspA was indeed successfully expressed at the cellsurface.

2.8.2 Expression and Surface Exposure of OspC

Despite poor expression in N. meningitidis (FIG. 8), cells carrying thefC9 construct surprisingly showed a signal with immunostaining (data notshown). This indicates that the N-terminal part of fHbp (as previouslydescribed in fA6) can be used for the surface localization of at leastC-terminal parts of OspC.

2.8.3 Expression and Surface Exposure of the Non-BorrelialNon-Lipoprotein RmpM

Both constructs (named fR1 and fTR1, see FIG. 11), were successfullyexpressed in E. coli and N. meningitidis.

Although immunostaining yielded bright signals for both constructs, thenegative control (N. meningitidis cells without construct) showed brightfluorescence as well. This indicated either a-specific binding of theMN2D6D monoclonal antibody to Neisseria cells, or unanticipated surfacelocalization of (parts of) RmpM. Therefore, a RmpM knockout was createdusing plasmid pCF13 (71). Plasmids carrying constructs fR1 and fTR1 weretransformed in this ARmpM strain as before and successful expression ofboth constructs was determined by Western blot (see FIG. 12).

Immunostaining of N. meningitidis cells and the RmpM knockout showedthat there was a-specific binding of the RmpM antibody at high antibodyconcentrations (strong staining of the knockout at 1:300 dilution andweak staining of the knockout at 1:2000 dilution, data not shown), whileat low antibody concentrations (1:10,000) staining of the knockoutcompletely vanished (data not shown). However, some normal Neisseriacells still showed staining at this low concentration (data not shown),indicating that at least for part of the cells RmpM might be (partially)surface exposed.

When placed in the RmpM knockout background, both fR1 and fTR1 showed afluorescent signal at the lowest antibody concentration (1:10,000),while staining was completely absent for the RmpM knockout withoutconstruct at this concentration (data not shown). This indicates thatthe N-terminal parts of both fHbp and TbpB that were previously used forthe surface localization of OspA can also be used for the surfacelocalization of an otherwise (predominantly) periplasmic non-lipoproteinof Neisseria meningitidis.

2.9 Immunogenicity of nOMVs Carrying OspA and fHbp-OspA Hybrids

Next, we investigated the sera of mice immunized with nOMVs carryingheterologous (fusion) proteins using Western blot. Pooled sera of all 21groups were blotted on membranes loaded with total protein content of E.coli with either pEN11-OspA or pEN11-Imp (see FIG. 3A for some of thesegroups). Pooled sera of the groups immunized with nOMVs carrying bothlow and high doses of constructs fA4b, fA4c, fA6, and fA7 all showed astrong band with the molecular mass of OspA (˜28 kDa), indicating astrong antibody response. Additionally, the pooled sera of the groupimmunized with the 20 μg/ml dose of OspA-carrying nOMVS showed a veryweak band at ˜28 kDa (data not shown). All other groups showed nodetectable response.

To see whether all individual mice raised OspA-specific antibodies, weblotted the sera of all ten individual mice from two highly responsivegroups (20 μg/ml fA4b and 20 μg/ml fA6). In all cases a ˜28 kDa band wasdetected (see FIG. 3B for the five mice immunized with 20 μg/ml fA6,data for fA4b not shown). This shows that the observed signals in thepooled sera were not due to single hyper-responders in the groups.

Note that the ˜36 kDa background signal in the pooled sera of the highdose fA6 group (FIG. 3A) is clearly caused by a single individual (mouse#4 in FIG. 4B).

In order quantify the immune response, sera of all individual miceimmunized with 20 μg/ml nOMVs carrying constructs fA4b, fA4c, fA6, orfA7 were tested for their binding capacity to purified OspA protein.Results for constructs fA4b and fA6 are shown in FIG. 4. The signaldetected for fA4b was ˜1.5 times higher than that observed for fA6 andfA7 and ˜2.5 times higher than that observed for fA4c (data not shown).

3. Discussion

OMVs are gaining attention as a robust and engineerable vaccine platformagainst many bacterial diseases. With the recognition of their vaccinepotential, interest in heterologous expression in OMVs has flourished.One of the unresolved issues regarding heterologous expression in OMVsis whether the location of the heterologous antigen (lumen, inside ofOM, outside of OM) affects the immune response that it evokes. For wholecells, some studies indicate that heterologous antigens are moreimmunogenic when they are located on the cell surface than when theyreside beneath it (59, 60), and it has been suggested that the same maybe true for OMVs (21). This makes the display of heterologous proteinson the OMV surface of special interest.

Here, we demonstrate a novel approach for the surface display ofheterologous antigens in OMVs. A detergent-free OMV extraction processwas recently developed for Neisseria that allows surface-exposedlipoproteins to remain attached to the OMV. This new extraction protocolopens up the possibility to decorate the surface of Neisseria OMVs withheterologous lipoproteins. We therefore expressed OspA, a Borrelialsurface lipoprotein, in N. meningitidis. Although OspA could be detectedin Neisseria cells and OMVs, we found no evidence for its surfaceexposure. We then constructed fusions between OspA and fHbp, ameningococcal surface lipoprotein. Several of these constructsconsisting of N-terminal parts of fHbp linked to OspA could be detectedat the cell surface using immunostaining. Furthermore, we havedemonstrated that technology is more broadly applicable as lipoproteinmediated surface expression could also be mediated by N-terminal partsof other lipoproteins, such as TbpB. In addition we demonstrated that avariety of antigenic proteins, including non-borrelial non-lipoproteinssuch as RmpM, could be surface expressed by fusion to N-terminal partsof lipoproteins. Hence, we have shown that the technology is broadlyapplicable to mediate the surface expression of specific antigens. Theantigens may be associated with an infectious disease and/or a tumour.The fusion proteins of the invention cause surface expression of suchantigens on e.g. OMVs, which triggers an immune response, resulting inthe prevention or treatment of an infectious disease or tumourassociated with the antigen.

In this respect, OMVs carrying these constructs elicited a strong immuneresponse in immunized mice, while OMVs carrying constructs that showedno evidence of surface exposure did not.

3.1 Outer Membrane Translocation

Knowledge of factors that govern the translocation of lipoproteins overthe OM is frugal and incomplete. In Borrelia, the lipoprotein tether(the unstructured region adjacent to the N-terminal cysteine) of severalsurface lipoproteins (OspA, OspC, and Vsp) seems to contain essentialinformation for OM translocation, since deletion or mutation of aminoacids in this region can lead to subsurface localization. Furthermore,fusion of the signal peptide and tether of these three lipoproteins tored fluorescent protein leads to surface localization of this otherwiseperiplasmic reporter-protein in Borrelia (47, 51, 55).

Neisseria has only a few surface lipoproteins and what factors affecttheir translocation over the OM is unknown. Our data indicate that, atleast for fHbp, these factors are different from those found inBorrelia.

In our hands, expression of OspA in N. meningitidis leads tomislocalization in the periplasm or periplasmic side of the outermembrane. This is in line with previous findings that expression of alipoprotein in an alternative host can lead to mislocalization, probablybecause host factors define the localization rules (51). We reasonedthat hybridization with an autologous surface exposed lipoprotein fromNeisseria like fHbp or TbpB might rescue this mislocalization andtherefore constructed fusions between OspA and different parts of fHbpor TbpB. Replacement of the OspA signal peptide and tether with that offHbp (construct fA1) did not restore OspA surface localization. However,when the fHbp part of this fusion gene was extended with the first 17N-terminal amino acids of the globular domain of fHbp (consisting of thefirst alpha-helix and subsequent loop), the resulting construct (fA6)could be detected at the cell surface. This indicates that sorting rulesmay differ between Neisseria and Borrelia, since in the former theregion affecting outer membrane translocation seems to extend beyond thetether region.

Almost all constructs that consists of fusion of OspA to an N-terminalpart of fHbp longer than that in fA6 were detected at the cell surface(fA4b, fA4c, fA7). The only exception is construct fA2b, which containsamino acids 1-152 of fHbp (the so-called N-terminal domain) coupled toOspA via an artificial linker. It is however important to stress thatconsidering the extremely poor expression level of fA2b in cells (seeFIG. 2A), it is not possible to discriminate whether the lack of signalafter immunostaining resulted from subsurface localization or poorexpression. We discuss this localization-expression problem furtherbelow. Constructs in which OspA was placed in between N-terminal andC-terminal parts of fHbp (fA3b, fA5c) were not detected at the cellsurface.

3.2 Effect of Antigen Location on Immunogenicity

We found evidence for surface exposure of constructs fA4b, fA4c, fA6,and fA7 in Neisseria cells. Coinciding with this, OMVs carrying theseconstructs were the only ones to elicit strong antibody responses inimmunized mice. This suggests that only antigens that are located on thesurface of the OMV can elicit antibody responses. However, it isimportant to realize that the expression level of the four ‘surface’constructs is clearly higher than that of the other constructs, both incells and nOMVs (see FIG. 2). Failure to detect a specific constructafter immunostaining could therefore also result from low expressionlevels rather than subsurface localization. It is not possible toexclude this scenario for constructs that have a very low expressionlevels in cells, e.g. fA1 and fA2b. However, cells that express OspA,fA3b, or fA5c have considerably higher expression levels, and it seemsunlikely that we would not observe them after immunostaining in casethey were surface exposed.

If we compare the Western blots of the groups of mice immunized withhigh dose OspA and low dose fA6 carrying OMVs (FIG. 2A, blots 4 and 5from left), the difference in apparent immunogenicity is much higherthan we would expect based on the observed expression levels of theseconstructs in OMVs (FIG. 2B). This indicates that surface display of aheterologous antigen in the OMV can indeed lead to enhancedimmunogenicity.

3.3 Vaccine Potential

Borrelia vaccines that are currently in development are subunit vaccinesthat are based on various recombinant lipidated OspA serotypes (38, 39).However, subunit vaccines suffer from poor immunogenicity and requirethe use of adjuvants. The presentation of OspA on the surface ofNeisserial nOMVs may result in better immunogenicity because of theintrinsic adjuvant activity of the nOMV and the presentation of theantigen in its native conformation.

Our OMV surface display method is generally applicable to otherantigenic proteins from pathogens, including also non-lipoproteins,especially since it has been shown that lipidation of non-lipoproteinsvia fusion to lipoproteins can enhance immunogenicity (62). Furthermoreit is possible to combine the expression and OMV surface display ofmultiple heterologous antigens in order to facilitate the production ofmultivalent heterologous OMVs.

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1-15. (canceled)
 16. A fusion lipoprotein comprising an N-terminal and aC-terminal fusion partner, wherein: a) the N-terminal fusion partnercomprises in N- to C-terminal order: i) a lipidated N-terminal cysteine;ii) a tether of a surface exposed lipoprotein of a Gram-negativebacterium; and, optionally, iii) a stretch of at least 5, 10, or 17contiguous amino acids that are located C-terminally of a tether in theamino acids sequence of a surface exposed lipoprotein of a Gram-negativebacterium; and wherein the N-terminal fusion partner causes expressionof the fusion lipoprotein on the extracellular outermembrane surface ofa Gram-negative bacterium upon expression therein; and, b) theC-terminal fusion partner comprises at least one epitope of an antigenassociated with an infectious disease and/or a tumour, and wherein theamino acid sequence of the fusion lipoprotein does not occur in nature.17. The fusion lipoprotein according to claim 16, wherein the tether islocated adjacent to the lipidated N-terminal cysteine.
 18. The fusionlipoprotein according to claim 16, wherein the N-terminal fusion partnercomprises an N-terminal fragment from a surface exposed lipoprotein of aGram-negative bacterium and wherein the fragment causes surfaceexpression of the fusion lipoprotein when expressed in the Gram-negativebacterium.
 19. The fusion lipoprotein according to claim 16, wherein theGram-negative bacterium is of the genus Neisseria.
 20. The fusionlipoprotein according to claim 18, wherein the surface exposedlipoprotein is selected from the group consisting of fHbp, LpbB, TbpB,NHBA and Ag473.
 21. The fusion lipoprotein according to claim 16,wherein the N-terminal fusion partner comprises at least one of: a) anamino acid sequence that has at least 60% sequence identity to the aminoacid sequence in positions 20-38 of SEQ ID NO: 1; b) an amino acidsequence that has at least 60% sequence identity to the amino acidsequence in positions 21-61 or positions 21-63 of SEQ ID NO: 2; or, c)an amino acid sequence that has at least 60% sequence identity to theamino acid sequence in positions 23-51 of SEQ ID NO:
 3. 22. The fusionlipoprotein according to claim 21, wherein: a) an amino acid sequencethat has at least 60% sequence identity to the amino acid sequence inpositions 20-50 of SEQ ID NO: 1; b) an amino acid sequence that has atleast 60% sequence identity to the amino acid sequence in positions21-73 or positions 21-75 of SEQ ID NO: 2; or, c) an amino acid sequencethat has at least 60% sequence identity to the amino acid sequence inpositions 23-63 of SEQ ID NO:
 3. 23. A fusion lipoprotein according toclaim 16, wherein the C-terminal fusion partner lacks amino acidsequences from the surface exposed lipoprotein from which the sequencesof the N-terminal fusion partner are derived.
 24. The fusion lipoproteinaccording to claim 23, wherein the C-terminal fusion partner comprisessurface exposed epitopes from a proteinaceous antigen of an infectiousagent or tumour.
 25. A fusion lipoprotein according to claim 24, whereinthe C-terminal fusion partner comprises a surface exposed domain of asurface exposed bacterial protein or lipoprotein.
 26. The fusionlipoprotein according to claim 25, wherein bacterial protein comprises aBorrelia surface lipoprotein selected from the group consisting of OspA,OspB, OspC, OspF, VlsE, BbCRASP1, Vspl, P35 (BBK32), P37 (BBK50), P39,P66, DpbA and BB017.
 27. The fusion lipoprotein according to claim 21,wherein the Borrelia surface lipoprotein comprises amino acids 29-273 ofSEQ ID NO: 4, amino acids 29-273 of SEQ ID NO: 58 or amino acids 136-210of SEQ ID NO:
 59. 28. An OMV comprising a fusion lipoprotein accordingto claim
 16. 29. The OMV according to claim 28, wherein the OMV is not adetergent-extracted OMV.
 30. The OMV according to claim 28, wherein theOMV is a supernatant OMV or a native OMV.
 31. The OMV according to claim28, wherein the OMV is obtainable from a Gram-negative bacterium thathas one or more genetic modifications selected from the group consistingof: a) a genetic modification causing the bacterium to produce an LPSwith reduced toxicity; b) genetic modification that increases vesicleformation; and, c) genetic modification that prevent proteolytic releaseof cell surface-exposed lipoprotein.
 32. The OMV according to claim 31,wherein the Gram-negative bacterium belongs to a genus selected from thegroup consisting of Neisseria, Bordetella, Escherichia and Salmonella.33. A pharmaceutical composition comprising an OMV according to claim 28and a pharmaceutically accepted excipient.
 34. A method of prevention ortreatment of an infectious disease or tumour associated with theantigen, comprising administering to a subject in need thereof an OMVaccording to claim
 21. 35. A nucleic acid molecule encoding apre-profusion lipoprotein, wherein, upon expression of the pre-profusionlipoprotein in a Gram-negative bacterium, the pre-profusion lipoproteinmatures into the fusion lipoprotein according to claim
 16. 36. AGram-negative bacterial host cell comprising a nucleic acid molecule oran expression construct according to claim 35.